Infectious Diseases – Baiyong Li, Yu Xia, Peng Zhang, Xinghua Pang, Zhongmin Wang, Akeso Biopharma Inc

Abstract for “Anti-CTLA4 antibodies”

“The present invention is related to tumor therapy and molecular immunelogy. This invention concerns an anti-CTLA4 anti-antibody, pharmaceutical composition, and its use. The anti-CTLA4 anti-antibody of the invention can bind CTLA4 and block CLTA4 from binding to B7.

Background for “Anti-CTLA4 antibodies”

“Cytotoxic T-lymphocyte associated antigen 4 (abbreviated CTLA4) shares a close relationship with CD28 in gene structure, sequence homology, gene expression, and chromosome location. Both receptors are for the co-stimulative mole B7 and are mainly expressed on activated T cells. CTLA4 can bind to B7 and inhibit activation of human and mouse T cells. It also plays a negative regulatory role in activation.

“CTLA4 Abs or CTLA4 Ligands can block CTLA4 binding to its nativeligands. This will prevent CTLA4’s transduction of the negative T cell regulating signal by CTLA4 as well as enhancing Tcells’ response to antigens. This is why in vivo as well as in vitro results are largely in agreement. There are currently several CTLA4 mAbs in clinical trials to treat prostate cancer, bladder cancer and colorectal carcinoma, as well as cancers of the gastrointestinal tract, liver, lung, and malignant melanoma. An overview of translational and preclinical research on CTLA-4 blockade. Grosso J F., Jure-Kunkel M N., Cancer Immun. 2013; 13:5. Epub 2013 January 22.

T cells produce “Interleukin 2” (IL-2). It regulates a subset of T cells and is a growth regulator. It also plays a key role in modulating the immune response. It is known to stimulate and activate the growth of B cells and can also be involved in antibody reaction, tumor surveillance, and hematopoiesis. The US FDA has approved recombinant human IL-2 for treatment of malignant tumors, including melanoma and kidney tumors. Clinical trials are also underway to treat chronic viral infections (Pharmacologic administrations of interleukin-2). Chavez, A. R., et al., Ann N Y Acad Sci, 2009. 1182: 14-27). Experiments in vitro have shown that CTLA4 mAbs can activate T cells and induce IL-2 production. This makes them a promising candidate for gene therapy against cancer and other diseases.

“As key factors in T cell function, CTLA4 or CTLA4-mAbs can have a specific therapeutic effect on certain diseases by interfering the immune microenvironment. They are highly effective and can be used to replace traditional medications. This opens up a new avenue for gene therapy. In clinical trials, experiments are ongoing to test CTLA4 and CTLA4-mAbs. They were shown to inhibit airway hyperresponsiveness in an animal model for asthma and prevent the development of rheumatic disorders. However, while biological gene therapy has not been shown to cause adverse effects in short-term clinical trials, it is important to consider the possible long-term consequences. Excessive blocking of CTLA4B7 signaling may lead to autoimmune diseases. Because antibodies can specifically bind their antigens, and cause the lysis or blocking of pathology, the development and use of drugs based upon antibodies is important for the treatment of malignant tumors in humans and other immune diseases.

“Inventors identified the hybridoma line LT002(CTLA4-4G10), and deposited it at China Center for Typical Culture Collection on Jun. 16th of June 2015 under the CCTCC No: C201587. Surprisingly, the inventors discovered that the hybridoma cell LT002 could secrete and produce a monoclonal antibody specific to binding CTLA4 (designated 4G10). This monoclonal antibody was capable of blocking the binding of CTLA4 and B7 with great effectiveness. The inventor also created several anti-CTLA4 humanized antibodies, including 4G10H3L3, 4,G10H4L3, and 4G10H5L3.

“The following inventions are available.”

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4. It may be a heavy-chain variable region or a light-chain variable region. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4, which comprises a heavy-chain variable region and/or light-chain variable regions. The heavy chain variable area comprises:

“In one embodiment, an antigen binding fragment of the antibody or antigen is composed of a heavy-chain variable region consisting of: an HCDR1 comprising SEQID NO: 21, an HCD2 comprising SEQID NO: 22 and an HCDR3 comprising SEQID NO: 23; and/or an LCDR1 comprising SEQID NO: 24, an LCR2 comprising SEQID NO: 25, an LCDR2 comprising SEQID NO: 25, and an LGDR3 comprising SEQID NO:26

“Another embodiment of the antigen binding fragment of the antibody or antigen is an HCDR1 that contains the amino acids sequence of the SEQID NO: 21 (wherein the X1=M) and an HCDR2 that comprises the amino sequence of the SEQID NO: 22 (wherein the X1=N, D, X2=T, or D, and X3=A, and X4=Q); and/or a light chain variable area consisting of the SEQID NO:26 and the X1=W, and the X2=SEQID NO:26 (wherein the X1=W, and the X1=M and X2=SEQID NO:26; wherein X1=W, and X1=T, respectively, X1=T, X2=T, X1=T, X1=A, X2=A, and X1=W, and the X2=W and the X2=T, respectively, Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 21 (wherein the X1=M), an HCD2 that contains the sequence of the SEQID NO: 22 (wherein the X1=N or D, X2=T and X4=Q), an HCDR2 that contains the sequence of the SEQID NO: 23, and an HCDR3R3 which has the sequence of the SEQID NO: 26 (wherein the X1=W) and the X2=W and the X1=W, wherein the X2=SEQID NO:26, with the X1=W, X1=W, X1=W, X1=W, X2=T and X1=T, respectively, wherein X1=K, X2=T and X2=A, and the X1=T, and X1=T and X1=W, X1=W, X2=W, X2=W, X2=SEQ ID:26 are X1=W, and X1=W, respectively, and X2=W, and X1=W, X1=S, X2=T and X1=T, and X2=T and X2=T and X2=SEQ IDNO:2=T and X1=W, and X1=SEQ ID:1=SEQ ID:1=W, and X1=T and X1=W, and X1=W, and X2=T and X1=W, respectively, and X2=W, and X1=W, and X1=W, and X1=W, and X1=A, and X2=T and X2=S, and X2=W, and X2=T and X2=T and X2=W, and X2=W, and X2=W, and X2=S, and X2=T and Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, an HCDR2 that contains the sequence of the SEQID NO: 29, and an HCDR3 which contains the sequence of the SEQID NO: 29. A light chain variable area may include an LCDR1 that has the sequence of the SEQID NO: 31 and an LCDR3 with the sequence of the SEQID NO: 32, and an LCDR3 which has the sequence of the SEQID NO:32 and the X1=W and X2=T and the SEQID NO:31 and the X1=W and the SEQID NO:31 and an LCDR2R2R2 = X1=T and the SEQID NO:36 Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, and an HCDR3 which contains the amino sequence of the SEQID NO: 29 (where X1=N, D, X2=T, or D); and/or a light-chain variable region consisting of an LCDR1 that includes the sequence of the sequence of the SEQID NO: 31 and an LCDR3 with the SEQID NO: 32 (where X1=W) and the sequence of the SEQID NO:32 Another embodiment of the antigen binding piece or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, and an HCDR3 which contains the amino sequence of the SEQID NO: 29 (where X1=N or D, X2=T, or D); and/or a light-chain variable region consisting of an LCDR1 that has the sequence of the SEQID NO: 31 and an LCDR2 with the SEQID NO: 32 (where X1=W, and X2=W and an LCDR3 with the sequence of the SEQID NO:32) and an LCDR3 including the SEQID NO:32 One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a FFab?, or an F(ab) A Fd, Fv, or a dAb can be used to select the antigen binding fragment of an antibody or antigen-binding fragment from a Fab, a F?, or an F(ab)2.

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4, and comprises a heavy-chain variable region and/or light-chain variable regions, wherein the heavy chains variable region includes SEQ ID No: 4. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light-chain variable regions, with the light chain variable area being SEQ ID No: 6. One embodiment of the invention includes an antigen binding or antibody fragment that binds human CTLA4. It consists of a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO: 4, while the light chain variable is SEQID NO: 6. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The heavy chain variable area comprises at minimum 90%, 95% and 96% respectively. The light chain variable regions comprises at least 90% and 96% respectively. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 4. and/or the light channel variable region comprises 1,2, 3, 4, 5, 7, 8, 7, 9, 9 or 98% of the variable light chains of SEQID NO: 6. Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 4 and SEQID NO: 6. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a F?, or a Fab. A Fd, Fv, or a dAb are all possible options.

“One embodiment of the invention contains an antigen binding fragment or antibody that binds human CTLA4, which includes a heavy-chain variable region and/or light-chain variable regions, wherein the heavy chains variable region includes SEQ ID No:8. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and/or light chain variable regions, with the light chain variable area being SEQ ID No:10. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It consists of a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:8 while the light chain variable is SEQID NO:10. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:8 and the light chain variable regional comprises SEQ ID No:10. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain chain contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID Nos: 8. and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, and/or the variable light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 10 amino Acid substitutions in SEQ ID Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 8 and SEQID NO:10. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a F?, or a Fab. A Fd, Fv, or a dAb are all possible options.

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area includes SEQ ID No:12. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light chain variable regions, with the light chain variable area being SEQ ID No:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:12, while the light chain variable regions comprises SEQID NO:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:12 and the light chain variable regional comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain chain region contains 1, 2, 3, 4, 6, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 12. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98% and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, amino acid substitutions within the variable lightchain of SEQID NO: Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 12 and SEQID NO: 14. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area is SEQ ID No:16. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions are SEQ ID No:16, and the light-chain variable region is SEQ ID no:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:16 and the light chain variable regional comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain region contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 16. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID ID NO. Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 16 and SEQID NO: 14. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4 and comprises a heavy-chain variable region and/or light chain variable regions, wherein the heavy-chain variable region is SEQ ID No:18. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:18, while the light chain variable area comprises SEQID NO:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The heavy chain variable area comprises SEQ ID No:18 and the light chain variable regions comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain region contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 18. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98%, and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, and/or the variable lightchain of SEQID NO: Some embodiments of the sequence variation and amino acid substitutions in SEQID NO: 18 or SEQID NO: 14 are not found in the CDR areas identified in SEQID NOS: 27-32. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable regions. The heavy chain variable area includes SEQ ID No: 19. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light-chain variable regions, with the light chain variable area being SEQ ID NO. 20. The invention contains an antigen binding or antibody fragment that binds human CTLA4, which is a combination of a heavy-chain variable region and a lighter chain variable area. In one embodiment, the heavy chain variable regions comprises SEQID NO: 19, while the light chain variable regions comprises SEQID NO: 20.

“In one embodiment, an antibody or antigen binding piece of the invention (described previously) is isolated.”

“In one embodiment, an antigen binding fragment or antibody of the invention (described previously) is produced in a cell CHO.”

“In one embodiment, an antigen binding fragment or antibody of the invention (described previously) optionally has at minimum one of the following characteristics. It detects CTLA4 levels in samples, crosses-reacts to cyno CTLA4, blocks CTLA4 binding to B7, regulates (e.g. down-regulates or regulates) CTLA4 activity or CTLA4, activates lymphocytes, and/or increases IFN-expression in lymphocytes. “in T lymphocytes.”

“In one embodiment, this invention relates to the hybridoma line LT002 deposited at China Center for Typical Culture Collection on Jun. 16, 2015, under CCTCC C201587.”

“In one embodiment, this invention relates to monoclonal antibodies produced by hybridoma cell lines LT002 and deposited at China Center for Typical Culture Collections (CCTCC), Jun. 16, 2015, under CCTCC C201587.

“The invention also covers isolated polypeptides containing the amino acid sequence of any of SEQ ID Nos. 4, 6, 8, 10, 12, 14 16, 18, 19, or 20.”

“The invention also refers to pharmaceutical compositions that contain any of the antigen binding fragments or antibodies of the invention. The invention may include any of the antigen binding fragments or antibodies of the invention. It also includes a pharmaceutically acceptable carrier/excipient. One embodiment of the invention includes any of the antigen binding fragments or antibodies of the invention and also contains a second therapeutic agent. An anti PD1 antibody, an antigen-binding fragment thereof, an anti -LAG3 antibody or antigen-binding fragment thereof, an anti TIM3 antibody or antigen-binding fragment thereof, an anti TIM3 antibody or antigen-binding fragment thereof and an anti ICOS antibody. One embodiment of the anti-PD1 antibody, or an antigen bound fragment thereof, is pembrolizumab.

“The invention also includes a conjugate containing an antigen binding fragment or antibody according to the invention, and a conjugated moiety. The conjugated moiety can be a detectable label in one embodiment. One embodiment of the conjugated moiety includes a radioisotope or fluorescent substance, an enzyme, a luminescent material, or a chromogenic substances.

“The invention also includes a kit that contains an antibody or antigen-binding fragment thereof according the invention or a conjugate according the invention and a second antigen which recognizes the antibody or antigen binding fraction thereof.” The second antibody may also include a detectable label such as a radioisotope or fluorescent substance, an enzyme, or a chromogenic material.

“The invention also includes nucleic acid molecules that encode the antigen binding fragments or antibodies of the invention or the polypeptides. The nucleic acid sequence SEQ ID NO. 3 is one embodiment of the invention. In another embodiment, it comprises the nucleic acids sequence of SEQID NO:5. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:7. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:9. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:11. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:13. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:15. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:17. The invention also includes vectors containing the nucleic acid sequence of SEQ ID NO:17 and host cells containing the nucleic acid sequence or vectors of invention.

“The invention also includes a method for producing an antigen or binding fragment. This involves: (i) cultivating a host cell with a polynucleotide that encodes the heavy chain or the light chain of any of the antigen binding pieces or antibodies of the invention under conditions conducive to expression; (ii) optionally recovering the antigen or binding fragment from the culture medium and/or host cell.”

The invention also includes a method for treating cancer in a person. This involves administering an effective amount anti-PD1 antibody or an antigen binding fragment thereof to the subject. An anti PD1 antibody, an antigen-binding fragment thereof, an anti VISTA antibody, an antigen-binding fragment thereof, an anti TIM3 antibody and an antigen-binding fragment thereof, an anti TIM3 antibody/an antigen-binding fragment thereof, an anti ICOS antibody/an antigen-binding fragment thereof or anti PDL1 antibody/an antigen-binding fragment thereof, an anti ILT4 antibody/an antigen-binding fragment thereof, an anti -CD73 antibody/an antigen-CD47 antibody/an antigen-CD47 or a fragment of the latter. One embodiment of the anti-PD1 antibodies or antigen binding pieces thereof can be selected from the following: pembrolizumab, an antigen bind fragment thereof, nivolumab, or an antigen bind fragment thereof.

The invention also includes a method for treating infection or other infectious diseases in humans. This involves administering an effective amount anti-PD1 antibody or an antigen binding fragment thereof to the subject. An anti PD1 antibody, an antigen-binding fragment thereof, an anti VISTA antibody, an antigen-binding fragment thereof, an anti TIM3 antibody and an antigen-binding fragment thereof, an anti TIM3 antibody/an antigen-binding fragment thereof, an anti ICOS antibody/an antigen-binding fragment thereof or anti PDL2 antibody; an anti ILT4 antibody; antigen-binding fragment thereof; anti ILT5 antibody; anti ILT5 or antigen-CD73 antibody; or antigen-binding fragment of the antigen-CD47 antibody; or antigen-binding fragment or antigen-binding fragment or antigen-binding fragment; or antigen-CD7 antibody; or antigen-CD47 antibody; or antigen-CD47 antibody; or antigen-binding fragment; or antigen-ILT5 or antigen-binding fragment or antigen-binding fragment or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or a-ILT5 or antigen-ILT5 or antigen-CD47; or if One embodiment of the anti-PD1 antibodies or antigen binding pieces thereof can be selected from the following: pembrolizumab, an antigen bind fragment thereof, nivolumab, or an antigen bind fragment thereof.

“The invention also includes a vaccine that contains an antigen binding fragment or antibody of the invention as well as an antigen.”

“The invention also includes a method of detecting the existence of a CTLA4 protein or a fragment thereof within a sample. This involves contacting the sample using an antibody or antigen binding piece of the invention, and then detecting the presence a complex between the fragment or the peptide. The detection of this complex signifies the presence CTLA4.

“The invention also includes a method for increasing the activity of an immuno cell. This involves contacting the immune cells with any of the antigen binding fragments or antibodies.”

“In one embodiment, the invention consists of a method to increase the activity of an immuno cell. This involves administering an effective amount antigen binding fragments or antibodies to the subject. This method can be used to treat cancer, an infection or infectious disease, or as a vaccine adjuvant.

“In one embodiment, an antigen or antibody binding fragment of the invention is used to prepare a medicament that can be used to increase immune cell activation, treat cancer, or treat infection or other infectious diseases.

“In one embodiment, the invention includes the use of an antigen binding fragment or antibody of the invention to make a medicament for treating cancer. This can be used for treating immune cell activation; cancer treatment; or infection or infectious disease.

“Another aspect of the invention is the use of the antigen binding fragment or antibody according to any of the embodiments to make a kit to detect the presence or amount of CTLA4 within a sample.”

“Another aspect of the invention is the use of the antigen binding fragment or antibody according to any of the embodiments or conjugates according to the invention in preparation of medicaments for the treatment or prevention of tumor, cancer, infection, or adjuvant therapy or diagnosis.”

“Another aspect of the invention is the use of the antibody/antigen binding fragment thereof according any one of the embodiments or the conjugate according the present invention in preparation of an agent that:

“Another aspect of the invention is the administration to cells of an effective amount antigen binding fragment or antibody according to any of the embodiments or conjugates according to the present inventor. The method can be selected from the following:

“Another aspect of this invention is a method for the prevention, treatment and/or adjuvant treatment and/or diagnosis a tumor or carcinoma. This involves administering to a subject an appropriate amount of the antigen binding fragment or antibody according to any of the embodiments or conjugates according to the invention.

“The antibody/antigen binding fragment of it according to any of the embodiments in the present invention for the prevention, treatment and/or adjuvant therapies and/or diagnosis a tumor/cancer.”

“The antibody/antigen binding fragment of it according to any of the embodiments in the present invention for use:

“Unless otherwise stated, technical and scientific terms used herein are the same as those who are skilled in the art. The most widely used methods in the relevant art include cell culture, molecular gene chemistry, and immunology. Below are definitions and explanations for relevant terms to help you better understand the invention.

“As used herein, when reference is made to the amino acid sequence of the CTLA4 protein (CytotoxicT-LymphocyteAntigen4), it includes the full length of the CTLA4 protein, or the extracellular fragment of CTLA4, CTLA4ECD (the portion of SEQ ID NO: 1 underlined with a wavy line), or a fragment comprising CTLA4ECD; it also includes a fusion protein of CTLA4ECD, e.g., the CTLA4ECD fragment fused to the Fc protein fragment of a mouse or human IgG (mFc or hFc) (see the description in Example 1). As those who are skilled in the art know, mutations or variations (including substitution, deletion, and/or addition) can be introduced to the CTLA4 protein’s amino acid sequence without affecting its biological function. The term “CTLA4 protein” is used in the present invention. All such sequences should be included, including the sequence underlined with an wavy line of SEQ ID No: 1, as well as the native and artificial variants. When a reference is made to the CTLA4 sequence fragment, it does not just include the sequence fragment of SEQID NO: 1, underlined with an wavy line but also the corresponding sequence fragments from its native or artificial variations.

“Sayned herein, except where otherwise stated, B7 refers only to B7-1 or B7-2. Their specific proteins sequences refers to sequences that are known to be in the art. You can refer to the sequences described in the literatures or GenBank.

“The term EC50, as used herein, refers to the concentration that causes 50% of the maximum effect.

“Antibody” is the term used herein. An immunoglobulin molecule that consists of two polypeptide chains, each with a?light? chain, is referred to as an antibody. A?heavy? and an L chain are two types of immunoglobulin molecules. (H) chain). You can classify antibody light chains as: Antibody light chains can be classified as? ???? light chain. You can classify heavy chains as??,?,?,????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????,??,??,? You can classify heavy chains as?,?,??,?,?????? or!, while the antibody type is IgM, IgD and IgG respectively. A?J? joins a light and heavy chain by joining a variable and constant regions. A region of approximately 12 or more amino acid is found in a light chain. The heavy chain also contains a?D?? A region of approximately 3 or more amino acid. Each heavy chain is composed of a heavy-chain variable region (VH), and a heavier chain constant region (CH). The heavy chain constant area consists of three domains (CH1,CH2 and CH3). Each light chain is composed of a light-chain variable region (VL), and a light-chain constant region (CL). The CL domain is the light chain constant region. The constant region of antibody mediates the binding of immunoglobulins to host tissues and factors. This includes various cells of immune system (e.g. effector cells) as well as the first component (C1q) of the classical complement system. The VH and VL regions are further subdivided into regions with high variability (referred as complementarity-determining region (CDR),) interspersed by regions known as framework regions (FR), which are more conserved. Each VH and VL is composed of 3CDRs or 4FRs. They are arranged in the following order, from the amino to the carboxy terminals: FR1, CDR1, CDR2, CDR2, CDR2, and FR3, CDR3, FR4, and FR5. Each pair of heavy and light chains has a variable region (VH) that forms an antigen binding spot. Each domain or region is assigned amino acids according to the Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health), Bethesda Md. (1987 and 1991), Chothia & Lesk (87) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342(888-883). “Antibody” is a generic term. The term?antibody? does not refer to any particular method of producing the antibody. It includes monoclonal, recombinant, and polyclonal antibody production methods. There are many types of antibodies, such as IgG (e.g. IgG1, IgG2, IgG3 subtype), IgA1, IgA2, IgD and IgE antibodies.

“Antigen binding fragment” is the term used herein. An antibody is a polypeptide that contains a fragment from a full-length antibody. This allows it to bind specifically to the antigen and compete with the full-length antibody to bind specifically to the antigen. It is also known as the?antigen binding section?. See Fundamental Immunology, Chapter. 7 (Paul W. ed. Second Edition Raven Press, N.Y. (1989),) which is incorporated by reference in its entirety herein for all purposes. Recombinant DNA techniques or chemical or enzyme cleavage can produce antigen binding fragments of antibody. In some cases, antigen binding fragments include Fab, Fab?, F(ab? ?2, Fd, Fv and dAb fragments, antigen binding fragments include Fab, Fab?, and F(ab)

“Fd fragment” is the term used herein. An antibody fragment containing the VH, CH1 and CH2 domains. The term ‘Fv fragment’? Refers to an antibody fragment that consists of the VL, VH domains of one arm of antibody. The term?dAbfragment? Refers to an antibody fragment that includes the VH domain (Ward and al. Nature 341:544-546 (1989); the term ‘Fab fragment? Refers to an antibody fragment that includes the VL, VH and CL domains. The term ‘F(ab)? )2 fragment? Refers to an antibody fragment that consists of two Fab fragments linked by disulfide bridges in the hinge region.

“In some cases the antigen binding fragment of an antibody is a single-chain antibody (e.g. scFv), in which the VL domain and VH domains pair up via a linker, which allows the production of a single-chain polypeptide chain to make a monovalent molecule (see, for example, Bird et., Science 242(4):423-426 (1988), and Huston et.l., Proc. Natl. Acad. Sci. USA 85:5879 -5883 (1988). Such scFv molecule can have the general structure: NH2?VL-Linker-VH-COOH or NH2?VH-Linker-VL-COOH. The repeated GGGGS amino acids sequence or its variants are suitable linkers in the prior art. A linker with the amino acid sequence (GGGGS4)4 may be used. However, its variants are also possible (Holliger and al. (1993)Proc. Natl. Acad. Sci. USA 90: 6444?6448 Alfthan et al. also describe other linkers that are useful in the invention. (1995) Protein Eng. 8:725-731; Choi et al. (2001) Eur. J. Immunol. 31: 94-106; Hu et al. (1996) Cancer Res. 56:3055-3061; Kipriyanov et al. (1999) J. Mol. Biol. 293:41-56 and Roovers et al. (2001) Cancer Immunol.”

“In certain cases, the antigen binding fraction of antibody is a monobody (a bivalent antibody), in which the VH domains and the VL domains are expressed on one polypeptide chain. The linker used is so short that two domains cannot pair with one another and must be paired with the complemental domain of another chain. This creates two antigen binding site (see Holliger P. and al., Proc. Natl. Acad. Sci. Sci.

“Antigen binding fragments (e.g. the above antibody fragments), can be obtained from given antibodies using conventional techniques which are well-known to those skilled in this art (e.g. recombinantDNA technique or enzymatic cleavage or chemical cleavage methods) and can be screened for specificity in much the same way as intact antibodies.

“Herein, except where otherwise indicated, includes intact antibodies as well as antigen binding fragments.

“As used herein the terms’mAb? “Molecular antibody” or “monoclonal antibodies?” An antibody or fragment of an antibody from a large number of homogenous antibody molecules. Monoclonal antibodies can be highly specific for a single epitope of the antigen. Polyclonal antibodies are more specific than monoclonal antibodies. They typically contain at least two or three antibodies that recognize different epitopes of the antigen. The hybridoma method, first described by Kohler and colleagues, is a good way to obtain monoclonal antibodies. (Nature 256:495,1975) or the recombinant-DNA technique (see U.S. Pat. No. No.

“Chimeric” is the term used herein. “Antibodies” refers to antibodies that are derived from antibodies (which can either be derived form a specific species or belong to a certain antibody class or class), while another part of the light and/or heavy chains is derived (which can also be derived (or derived) from an identical species or belonging an identical or differing antibody class or class), so long as they still have the ability to bind to the target antibody (U.S. Pat. No. Cabilly et al. ; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).”

“As used herein the term “humanized” means: “Antibodies” refers to antibodies or fragments of antibodies obtained by replacing any or all of the CDRs from a human immunoglobulin (recipient antibodies) with CDRs from a nonhuman antibody (donor antibodies). The donor antibody can be either a mouse, rat, or rabbit antibody that exhibits the desired specificity and affinity. To further enhance or optimize the performance of an antibody, it is possible to replace some of the amino acids in the recipient antibody’s framework regions (FRs), with the corresponding amino acids of the non-human antibody. For more details about humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2593-596 (1992); Clark, Immunol. Today 21: 397-402 (2000).

“Epitope” is the term used herein. Refers to the portion of an antigen that is bound specifically by an antibody or immunoglobulin. The art term “epitope” is used to refer to the part of an antigen that has been bound by an antibody or immunoglobulin. is also known as?antigenic determinaant?

An epitope, or antigenic determinant, generally consists only of the active surface group of the molecule. These include amino acids, carbohydrate compounds, or sugar side chains. They also have specific three-dimensional structural characteristics, and specific charge characteristics. An epitope can be composed of at least 3, 4, 5, 6/7, 8, 9, 10, 11, 12, 13 or 14 consecutive or inconsecutive amino acids in a far spatial conformation. It could be a ‘linear? It can be a?linear? epitope. Epitope Mapping Protocols In Methods in Molecular Biology Vol. 66, G. E. Morris, Ed. (1996). A linear epitope is a structure that contains all points of interaction between the protein (e.g., antibodies) along a primary amino acid sequence. Conformational epitopes have the inter-interacting points as they span the amino acid residues that are distinct from one another.

“Isolated anti-CTLA4 antibodies, antigen-binding pieces thereof and methods for their use are all included in the present invention. Antibody or antigen binding fragments are at least partially free from other biological molecules in the cells or cell culture where they were produced. These biological molecules can include nucleic acid, proteins, lipids and carbohydrates. A fragment of an antigen-binding antibody or other protein may be isolated from expression system components, such as biological molecules that are present in a host cell or the growth medium. The term “isolated” is generally used to refer to a cell that has been isolated from other cells. The term “isolated” does not refer to complete absence of these biological molecules, water, buffers or salts, or components of a pharmaceutical formula that include the antibodies or fragments.

“?Isolated nucleic acids molecule?” “?Isolated nucleic acid molecule” or “?isolated mononucleotide?” A DNA orRNA of genomic, molecular, or synthetic origin, which is not associated or linked to any polynucleotide. This disclosure should clarify that “a nucleic acid molecular comprising” does not include any nucleic acids. A particular sequence of nucleotides does not include intact chromosomes. Isolated nucleic acids molecules? Specific nucleic acids sequences can include, in addition, the specified sequences. Coding sequences for upto ten or more proteins, portions, or fragments thereof. Or may include operably linked regulatory sequencings that control expression.

“As used herein the term?E. What is the coli expression system? An expression system that consists of E.coli (strain), vector, and where E.coli (strain), is derived from commercial strains, such as GI698, E.2656, E.321, E.322, E.323, E.324, E.324, and E.324.

“Vector” is the term used herein. A nucleic acid carrying instrument into which a single polynucleotide may be inserted. Expression vectors are those that enable the expression of the protein encoded in the inserted polynucleotide. The vector can be introduced to a host cell through transformation, transduction, or transfection so that the gene component carried by the vector is expressed within the host cell. The art of vectors is well-known to those who are skilled in it. They include plasmid, cosmid, and artificial chromosomes, such as yeast artificial chromosomes (YAC), bacteria artificial chromosomes (BAC), or P1-derived artificial Chromosomes (PAC); bacteriophage (e.g.?). phage, M13 phage, and animal virus. Retroviruses, adenoviruses, adenoviruses, herpes viruses (e.g. herpes simplex virus), poxviruses, baculoviruses, papilloma and papova viruses (e.g. SV40) are all examples of animal viruses that can be used to create vectors. A vector may contain several components that control the expression of the gene, such as a promoter sequence and transcription initiation sequence, enhancer sequencing, selection component, reporter gene, and selector sequence. A vector may also contain a replication site.

“The term “host cell” is used herein. “Host cell” refers to any cell that can be used to introduce a vector. This includes prokaryotic and fungal cells like E. coli and Bacillus subtilis, as well as insect cells such S2 Drosophila cells or Sf9 as well as animal cells such fibroblast, CHOcells, COScells, NSOcells, HeLacells, BHKcells, HEK293cells, human cells.

“Specific binding” is the term used herein. The non-random binding reaction that occurs between two molecules, such a reaction between an antibody or its targeted antigen, is called?specific binding. An antibody that binds an antigen specifically (or an antibody specific to an antigen) in some embodiments means that the affinity (KD), of the antibody, is less than 10?5 M.

“KD” is the term used herein. The dissociation equilibrium of an antibody-antigen interaction is what’s used herein. It describes the binding affinity between the antigen and the antibody. The closer the antibody-antigen interaction is to the antigen, the greater the affinity between them. An antibody binds to the antigen when its dissociation constant (KD), is less than 10?5 M. This could be determined by surface plasmon resonance (SPR), on a BIACORE instrument, or a comparable technique (e.g. OCTET and KINEXA.

“As used herein the terms’monoclonal antibodies? “Monoclonal antibody” and “mAb?” are interchangeable terms. Both terms have the same meaning and can be interchangeably. The terms?polyclonal antibodies? are also interchangeable. The terms?polyclonal antibody? and?pub? have the same meanings. Both terms have the same meaning and can be interchangeably. The terms?polypeptide and?protein? are interchangeable. The terms?polypeptide? and?protein? have the same meanings. Both terms have the same meaning and can be interchangeably used. The present invention generally uses single-letter and three-letter abbreviations to represent amino acids. Alanine, for example, can be represented as Ala or A.

“The terms “hybridoma” and “hybridoma cell line” are used herein. Hybridoma cell line and?hybridoma? They can be interchangeable. When the term “hybridoma” is used, it also includes the subclonal and descendent cells of the hybridoma. It also includes the subclonal cells and descendent cells of hybridoma. When LT002 and LT003 are mentioned, it also includes the subclonal cells and descendent cells from the hybridoma line LT002 and LT003.

“Pharmaceutically acceptable carrier/excipient” is the term used herein. The term “pharmaceutically acceptable carrier and/or excipient” refers to a vector and/or an excipient compatible with the subject and the active part in pharmacology/physiology. These terms are well-known in the art (see Remington’s Pharmaceutical Sciences). Gennaro A. R., 19th edition. Pennsylvania: Mack Publishing Company 1995). They include, but are not limited to, pH adjusting agent and surfactant as well as adjuvant and ionic intens enhancer. The pH adjusting agent, for example, includes phosphate buffer. Surfactant can include anionic, cationic or nonionic surfactants, such as Tween 80, and ionic intensifier includes sodium chloride.

“Adjuvant” is the term used herein. “Adjuvant” is a non-specific immune enhancer that can increase or alter the immune response to an antigen. It may be delivered in combination with the antigen or before it enters the body. There are many adjuvants available, including aluminum adjuvant, such as aluminum hydroxide, Freund’s adjuvant, Corynebacterium Parvum, lipopolysaccharide and cytokine. The most widely used adjuvant in animal experiments is Freund’s, while aluminum hydroxide is used in clinical trials.

“Effective amount” is the term used herein. The effective amount is the amount that can achieve or at least partially achieve desired effects. Prophylactically effective amounts for diseases (e.g. a tumor or disease associated with CTLA4 activation or excessive binding to CTLA4) refer to an amount that is sufficient to stop, arrest or delay the development or progression of the disease. Therapeutically effective amounts for diseases refer to the amount that can cure the disease or at least partially stop its complications for the patient. This is something that can be done by those who are skilled in the art. A therapeutically effective amount will, for example, depend on the severity and condition of the disease, the general state of the immune system, the general health of the patient, and other factors such as age, body mass, weight, administration mode, and other therapies that are being administered simultaneously.

“As used in this document, including the appended Claims, the singular forms words such as ‘a,? ?an,? If the context is clear,?an? and?the? should be used together.

“?Administration? “?Administration” refers to any contact of an exogenous therapeutic, diagnostic, or pharmaceutical agent or composition with an animal, human subject, cell tissue, organ or animal.

“?Treat? “?Treat? The act of administering a therapeutic agent (e.g., a composition containing one of the antigen-binding fragments or antibodies of the invention) to an individual or subject suffering from one or more of the diseases or suspected of developing one, or both, of these symptoms. The agent is usually administered in a dose that is effective in relieving one or more symptoms of a disease in the subject or population. This may be done by either inducing or inhibiting the progression of the symptom(s). The effectiveness of any therapeutic agent to relieve a particular disease symptom will vary depending on factors like the age and weight of patients and the drug’s ability to induce a desired response. Any clinical measurement that is used by doctors or other skilled healthcare providers can help determine if a disease symptom has been relieved.

“Antibodies of the Invention.”

Monoclonal antibodies (MAB), which are therapeutic antibodies, have shown remarkable efficacy in treating a variety of diseases. The traditional way to obtain therapeutic antibodies is to immunize an animal with an antigen and then obtain antibodies against that antigen from the animal. Optionally, affinity maturation can be used to improve an antibody with low affinity to the antigen. This method can be labor-intensive and time-consuming. It also fails to pinpoint a specific epitope of the antigen.

“Antigen binding depends on the variable regions in the light and heavy chains; the variable regions of each chain comprise three hypervariable areas, also known as complementarity determining regions (CDR). The heavy chain (H), which includes HCDR1, HCD2 and HCDR3, while the light chain (L), comprises LCDR1, LCDR2 or LCDR3. For definitions, refer to Kabat et al. Sequences Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 93-3282, Bethesda MD

“The invention is in part related to anti-CTLA4 antibody sequences. The invention’s antibodies can specifically bind CTLA4. They are able to block CLTA4 from B7 binding, relieve CTLA4-induced immunosuppression and activate T lymphocytes.

“In particular, this invention refers to the mouse antibody 4G10 described herein. It comprises the heavy chain variable area (VH) and the light-chain variable region (VL), both of which are SEQ ID No:4; and humanized variants of this antibody.

“In certain embodiments, the humanized VH-sequences of the 4G10 antibody can include any of the following VH areas:

“In certain embodiments, the humanized VH sequence for the 4G10 antibody may include the following CDR areas (determined according to Kabat:”

“In alternate embodiments, the humanized VH sequence for the 4G10 antibody may include the following CDR areas (determined using VBASE2 database analysis:”

“In certain embodiments, the 4G10 antibody’s humanized VL sequences can include any of the following VL areas:

“In certain embodiments, the humanized sequence of the 4G10 antibody can include the following CDR areas (which were determined according to Kabat:

“In alternative embodiments, a humanized VL sequence for the 4G10 antibody may include the following CDR areas (which CDR areas were determined using VBASE database analysis).

“Any of these humanized VH regions can be paired with any one of the VL regions. Preferential embodiments include an antibody that includes the VH region in SEQ ID No: 19 and VL region in SEQ ID No: 20. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 8. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 12. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 16 and 14. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 18 and SEQID NO: 14.

“Antibody Expression”

“The invention’s antibodies and fragments can be made by any skilled person in the art. The invention’s antibodies and antibody fragments can be made in any cell. Eukaryotic and prokaryotic cells can be used as hosts to express the antibodies or fragments of immunoglobulin chains. This includes mammalian cells. These cells include, among others, Chinese hamster eggs (CHO), NSO, SP2 and HeLa cells. They also include baby hamster kidney (BHK), monkey kidney (COS) cells and human hepatocellular carcinoma (e.g. Hep G2) cells. Mammalian host cell types include mammalian human, mouse and rat cells, as well as bovine, horse, and hamster cells. Selection of cell lines that express high levels of expression is a way to identify cell lines of preference. Sf9 cells, plant cell lines, and amphibian cell lines are all possible cell types. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia ptjperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp. any Saccharomyces sp. Hansenula polymorpha any Kluyveromyces sp. Candida albicans any Aspergillus sp. Trichoderma reesei Chrysosporium luckynowense, Fusarium sp. Yarrowia crassa Recombinant expression vectors that encode the antigen-binding or heavy chain portion or fragment, and the light chain or antigen binding fragment thereof are introduced to mammalian host cell. The antibodies are created by cultivating the host cells for sufficient time to permit the expression or secretion of the antigen or fragment in the host cell or into the culture medium in order to produce them.

“Antibody Purification”

Standard protein purification methods can be used to recover antibodies and antigen binding fragments and immunoglobulin chain from culture media. There are many techniques that can be used to increase the expression of antigen-binding and antibody fragments and immunoglobulin chain of the invention or other moieties from cell lines. The GS system, which is a common method of increasing expression in certain circumstances, is an example. European Patent Nos. are discussed in part or whole. 0 216 846; 0 256 5055, 0 323 997, and European Patent Application No. 89303964.4. In one embodiment of the invention, mammalian host cells (e.g. CHO) lack a glutamine synetase genes and are grown in medium without glutamine. However, the polynucleotide encoding immunoglobulin chains contains a glutamine synthetase genetic which is complementary to the gene’s absence in the host cell.

The present invention provides methods for purifying an antigen-binding or antibody fragment of the invention. These methods include introducing the sample containing the antibody or fragment to a purification media (e.g. anion exchange medium or cation exchange medium), and then either collecting the purified antibody or fragment or discarding the flow through fraction. Finally, eluting the bound antibody from the medium and collecting the liquid eluate. The medium is contained in the column to which the sample is being applied. The purification process is performed according to an embodiment of the invention. For example, the medium is placed in a column to which the sample is applied.

“Antibody Engineering”

“In some embodiments, antigen-binding fragments and antibodies of the invention can be modified to enhance their properties by including modifications to the framework and/or CDRs. These engineered modifications can be made using molecular modeling. To understand the structure of the antibody, a molecular model can be built for the variable region in the non-human parental antibody sequence. This can then be used to identify regions that could interact with the antigen. Conventional CDRs are built on the alignment of immunoglobulins sequences and identification of variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al. ; National Institutes of Health Bethesda (Md. ; 5th ed. NIH Publ. No. No. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and her coworkers examined the conformations of loops in crystal structures for antibodies and suggested hypervariable loops. Chothia, et al., (1987) J. Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are differences between the?CDRs? regions. These include?hypervariable loops’ and?CDRs?. Later studies (Raghunathan et al, (2012) J Mol Recog. 25, 3, 103-113) Analyzing several antibody?antigen complexes, we found that antigen binding areas in antibodies don’t always conform to the?CDR. Antigen binding regions in antibodies do not always conform to the?CDR?. loops. To guide the selection of potential binding regions to the antigen, the molecular model of the non-human antibody variable region can be used. The model-based potential binding regions of antigen differ from conventional?CDRs or ‘hyper variable? in practice. loops. For molecular modeling, commercial scientific software like MOE (Chemical Computing Group), can be used. The best matches between the human frameworks and the non-human sequence can be used to select the human frameworks. The VJ regions of the human germlines that are part of FR4 (framework 4 in VH) are compared to the non-human counterpart. For FR4 in VL, JKappa and JLambda are compared to the corresponding region of the non-human germline sequences. After identifying suitable human frameworks, the CDRs can be grafted into those frameworks. Some residues at the VL-VH interface may be retained in some cases. For identifying potential CDR conformations that could alter and binding to antigen, Molecular models are also useful. These residues may be retained in their non-human (parental sequence) form. You can also use molecular models to identify solvent-exposed amino acids that could cause undesirable effects like glycosylation or deamidation. These potential problems can be eliminated or minimized by using developability filters.

“Another method of modifying the framework is to modify one or more residues in the framework region or within one or several CDR regions to remove T cell epitopes and reduce the antibody’s immunogenicity. This is sometimes called “deimmunization”. This approach is also known as?deimmunization?. No. 7,125,689.”

“In certain embodiments, it may be possible to alter some amino acids that have exposed side-chains to another residue to increase the chemical stability of final antibodies. This will avoid isomerization or deamidation. Asparagine can be deamidated on NG or DG. NG, NS and NA sequences. This creates an isoaspartic acids residue which introduces a kink in the polypeptide chain and decreases its stability. Isomerization can occur at DG or DS sequences. The antibodies described in the present disclosure may not contain asparagine or deamidation isomerism sites.

“For example, an Asparagine (Asn), residue can be changed to Gln, or Ala, to decrease the possibility of formation isoaspartate at Asn-Gly sequences. This is especially true for a CDR. Similar problems could occur in an Asp-Gly sequence. Reissner & Aswad, 2003 Cell. Mol. Life Sci. 60:1281. The formation of isoaspartate may cause the binding of an antibody against its target antigen to be disrupted or even broken. Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. One embodiment changes the asparagine to glutamine (Gln). To reduce the risk of deamidation (which occurs more frequently when small amino acids are near asparagine and glutamine), it may be possible to change an amino acid that is adjacent to an asparagine residue (Asn). See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. 662:261. Id. Id.

“Exemplary Stabilizing CDR Type Variants”

“CDR Residue Managing Variant Sequence”

“Antibody Engineering of the Fe Region.”

“Antibodies (e.g. humanized antibodies) can be modified to alter the Fc region. This is typically done to alter one or more properties, such as serum half life, complement fixation and Fc receptor binding and/or effector functions (e.g. antigen-dependent cell cytotoxicity). The antigen-binding pieces and antibodies described herein can also be chemically modified. For example, one or several chemical moieties can attach to the antibody, or modified to alter its glycosylation. This can alter any or all of the fragment’s properties.

Summary for “Anti-CTLA4 antibodies”

“Cytotoxic T-lymphocyte associated antigen 4 (abbreviated CTLA4) shares a close relationship with CD28 in gene structure, sequence homology, gene expression, and chromosome location. Both receptors are for the co-stimulative mole B7 and are mainly expressed on activated T cells. CTLA4 can bind to B7 and inhibit activation of human and mouse T cells. It also plays a negative regulatory role in activation.

“CTLA4 Abs or CTLA4 Ligands can block CTLA4 binding to its nativeligands. This will prevent CTLA4’s transduction of the negative T cell regulating signal by CTLA4 as well as enhancing Tcells’ response to antigens. This is why in vivo as well as in vitro results are largely in agreement. There are currently several CTLA4 mAbs in clinical trials to treat prostate cancer, bladder cancer and colorectal carcinoma, as well as cancers of the gastrointestinal tract, liver, lung, and malignant melanoma. An overview of translational and preclinical research on CTLA-4 blockade. Grosso J F., Jure-Kunkel M N., Cancer Immun. 2013; 13:5. Epub 2013 January 22.

T cells produce “Interleukin 2” (IL-2). It regulates a subset of T cells and is a growth regulator. It also plays a key role in modulating the immune response. It is known to stimulate and activate the growth of B cells and can also be involved in antibody reaction, tumor surveillance, and hematopoiesis. The US FDA has approved recombinant human IL-2 for treatment of malignant tumors, including melanoma and kidney tumors. Clinical trials are also underway to treat chronic viral infections (Pharmacologic administrations of interleukin-2). Chavez, A. R., et al., Ann N Y Acad Sci, 2009. 1182: 14-27). Experiments in vitro have shown that CTLA4 mAbs can activate T cells and induce IL-2 production. This makes them a promising candidate for gene therapy against cancer and other diseases.

“As key factors in T cell function, CTLA4 or CTLA4-mAbs can have a specific therapeutic effect on certain diseases by interfering the immune microenvironment. They are highly effective and can be used to replace traditional medications. This opens up a new avenue for gene therapy. In clinical trials, experiments are ongoing to test CTLA4 and CTLA4-mAbs. They were shown to inhibit airway hyperresponsiveness in an animal model for asthma and prevent the development of rheumatic disorders. However, while biological gene therapy has not been shown to cause adverse effects in short-term clinical trials, it is important to consider the possible long-term consequences. Excessive blocking of CTLA4B7 signaling may lead to autoimmune diseases. Because antibodies can specifically bind their antigens, and cause the lysis or blocking of pathology, the development and use of drugs based upon antibodies is important for the treatment of malignant tumors in humans and other immune diseases.

“Inventors identified the hybridoma line LT002(CTLA4-4G10), and deposited it at China Center for Typical Culture Collection on Jun. 16th of June 2015 under the CCTCC No: C201587. Surprisingly, the inventors discovered that the hybridoma cell LT002 could secrete and produce a monoclonal antibody specific to binding CTLA4 (designated 4G10). This monoclonal antibody was capable of blocking the binding of CTLA4 and B7 with great effectiveness. The inventor also created several anti-CTLA4 humanized antibodies, including 4G10H3L3, 4,G10H4L3, and 4G10H5L3.

“The following inventions are available.”

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4. It may be a heavy-chain variable region or a light-chain variable region. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4, which comprises a heavy-chain variable region and/or light-chain variable regions. The heavy chain variable area comprises:

“In one embodiment, an antigen binding fragment of the antibody or antigen is composed of a heavy-chain variable region consisting of: an HCDR1 comprising SEQID NO: 21, an HCD2 comprising SEQID NO: 22 and an HCDR3 comprising SEQID NO: 23; and/or an LCDR1 comprising SEQID NO: 24, an LCR2 comprising SEQID NO: 25, an LCDR2 comprising SEQID NO: 25, and an LGDR3 comprising SEQID NO:26

“Another embodiment of the antigen binding fragment of the antibody or antigen is an HCDR1 that contains the amino acids sequence of the SEQID NO: 21 (wherein the X1=M) and an HCDR2 that comprises the amino sequence of the SEQID NO: 22 (wherein the X1=N, D, X2=T, or D, and X3=A, and X4=Q); and/or a light chain variable area consisting of the SEQID NO:26 and the X1=W, and the X2=SEQID NO:26 (wherein the X1=W, and the X1=M and X2=SEQID NO:26; wherein X1=W, and X1=T, respectively, X1=T, X2=T, X1=T, X1=A, X2=A, and X1=W, and the X2=W and the X2=T, respectively, Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 21 (wherein the X1=M), an HCD2 that contains the sequence of the SEQID NO: 22 (wherein the X1=N or D, X2=T and X4=Q), an HCDR2 that contains the sequence of the SEQID NO: 23, and an HCDR3R3 which has the sequence of the SEQID NO: 26 (wherein the X1=W) and the X2=W and the X1=W, wherein the X2=SEQID NO:26, with the X1=W, X1=W, X1=W, X1=W, X2=T and X1=T, respectively, wherein X1=K, X2=T and X2=A, and the X1=T, and X1=T and X1=W, X1=W, X2=W, X2=W, X2=SEQ ID:26 are X1=W, and X1=W, respectively, and X2=W, and X1=W, X1=S, X2=T and X1=T, and X2=T and X2=T and X2=SEQ IDNO:2=T and X1=W, and X1=SEQ ID:1=SEQ ID:1=W, and X1=T and X1=W, and X1=W, and X2=T and X1=W, respectively, and X2=W, and X1=W, and X1=W, and X1=W, and X1=A, and X2=T and X2=S, and X2=W, and X2=T and X2=T and X2=W, and X2=W, and X2=W, and X2=S, and X2=T and Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, an HCDR2 that contains the sequence of the SEQID NO: 29, and an HCDR3 which contains the sequence of the SEQID NO: 29. A light chain variable area may include an LCDR1 that has the sequence of the SEQID NO: 31 and an LCDR3 with the sequence of the SEQID NO: 32, and an LCDR3 which has the sequence of the SEQID NO:32 and the X1=W and X2=T and the SEQID NO:31 and the X1=W and the SEQID NO:31 and an LCDR2R2R2 = X1=T and the SEQID NO:36 Another embodiment of the antigen binding fragment or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, and an HCDR3 which contains the amino sequence of the SEQID NO: 29 (where X1=N, D, X2=T, or D); and/or a light-chain variable region consisting of an LCDR1 that includes the sequence of the sequence of the SEQID NO: 31 and an LCDR3 with the SEQID NO: 32 (where X1=W) and the sequence of the SEQID NO:32 Another embodiment of the antigen binding piece or antibody comprises a heavy-chain variable region. This includes an HCDR1 that contains the amino acids sequence of the SEQID NO: 27, an HCD2 that contains the sequence of the SEQID NO: 28, and an HCDR3 which contains the amino sequence of the SEQID NO: 29 (where X1=N or D, X2=T, or D); and/or a light-chain variable region consisting of an LCDR1 that has the sequence of the SEQID NO: 31 and an LCDR2 with the SEQID NO: 32 (where X1=W, and X2=W and an LCDR3 with the sequence of the SEQID NO:32) and an LCDR3 including the SEQID NO:32 One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a FFab?, or an F(ab) A Fd, Fv, or a dAb can be used to select the antigen binding fragment of an antibody or antigen-binding fragment from a Fab, a F?, or an F(ab)2.

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4, and comprises a heavy-chain variable region and/or light-chain variable regions, wherein the heavy chains variable region includes SEQ ID No: 4. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light-chain variable regions, with the light chain variable area being SEQ ID No: 6. One embodiment of the invention includes an antigen binding or antibody fragment that binds human CTLA4. It consists of a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO: 4, while the light chain variable is SEQID NO: 6. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The heavy chain variable area comprises at minimum 90%, 95% and 96% respectively. The light chain variable regions comprises at least 90% and 96% respectively. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 4. and/or the light channel variable region comprises 1,2, 3, 4, 5, 7, 8, 7, 9, 9 or 98% of the variable light chains of SEQID NO: 6. Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 4 and SEQID NO: 6. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a F?, or a Fab. A Fd, Fv, or a dAb are all possible options.

“One embodiment of the invention contains an antigen binding fragment or antibody that binds human CTLA4, which includes a heavy-chain variable region and/or light-chain variable regions, wherein the heavy chains variable region includes SEQ ID No:8. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and/or light chain variable regions, with the light chain variable area being SEQ ID No:10. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It consists of a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:8 while the light chain variable is SEQID NO:10. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:8 and the light chain variable regional comprises SEQ ID No:10. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain chain contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID Nos: 8. and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, and/or the variable light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 10 amino Acid substitutions in SEQ ID Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 8 and SEQID NO:10. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from a F(ab), a F?, or a Fab. A Fd, Fv, or a dAb are all possible options.

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area includes SEQ ID No:12. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light chain variable regions, with the light chain variable area being SEQ ID No:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:12, while the light chain variable regions comprises SEQID NO:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:12 and the light chain variable regional comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain chain region contains 1, 2, 3, 4, 6, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 12. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98% and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, amino acid substitutions within the variable lightchain of SEQID NO: Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 12 and SEQID NO: 14. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area is SEQ ID No:16. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions are SEQ ID No:16, and the light-chain variable region is SEQ ID no:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light chain variable regions. The heavy chain variable area comprises SEQ ID No:16 and the light chain variable regional comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain region contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 16. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID ID NO. Some embodiments allow sequence variation or amino acids substitutions to occur in SEQID NO: 16 and SEQID NO: 14. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding or antibody fragment of the invention binds to human CTLA4 and comprises a heavy-chain variable region and/or light chain variable regions, wherein the heavy-chain variable region is SEQ ID No:18. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4, which includes a heavy-chain variable region and a lighter chain variable area. The heavy chain variable regions comprises SEQID NO:18, while the light chain variable area comprises SEQID NO:14. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The heavy chain variable area comprises SEQ ID No:18 and the light chain variable regions comprises SEQ ID no:14. One embodiment of the invention includes an antigen binding fragment or antibody that binds human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable region. The variable heavy chain region contains 1, 2, 3, 4, 6, 7, 7, 8, 9, 9 or 10 amino acids substitutions in SEQ ID No: 18. The light chain variable area comprises 1, 2, 4, 5, 6, 7, 7, 8, 9, 9 or 98%, and/or the variable light chains variable region 1, 2, 3, 5, 6, 7, 7, 8, 9, 9 or 10, and/or the variable lightchain of SEQID NO: Some embodiments of the sequence variation and amino acid substitutions in SEQID NO: 18 or SEQID NO: 14 are not found in the CDR areas identified in SEQID NOS: 27-32. One embodiment of the antigen binding fragment or antibody is a humanized antibody that includes two heavy chains and two light chain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG1 constantdomain. One embodiment of the antigen binding or antibody fragment is a humanized antibody with a human IgG2 constantdomain. One embodiment of the antigen binding fragment or antibody is a humanized antibody with a human IgG4 constantdomain. Another embodiment of the antigen binding or antibody fragment is a humanized antibody that has a human IgG1 constantdomain and a human Kappa constantdomain. Another embodiment selects the antigen binding fragment or antibody from an F(ab), an F?, and an Fab. )2, Fd, Fv, an Fv or an F(ab?).

“In one embodiment, an antigen binding fragment or antibody of the invention binds to human CTLA4. It comprises a heavy-chain variable region and/or light-chain variable regions. The heavy chain variable area includes SEQ ID No: 19. One embodiment of the invention contains an antigen binding or antibody fragment that binds human CTLA4. It includes a heavy-chain variable region and/or light-chain variable regions, with the light chain variable area being SEQ ID NO. 20. The invention contains an antigen binding or antibody fragment that binds human CTLA4, which is a combination of a heavy-chain variable region and a lighter chain variable area. In one embodiment, the heavy chain variable regions comprises SEQID NO: 19, while the light chain variable regions comprises SEQID NO: 20.

“In one embodiment, an antibody or antigen binding piece of the invention (described previously) is isolated.”

“In one embodiment, an antigen binding fragment or antibody of the invention (described previously) is produced in a cell CHO.”

“In one embodiment, an antigen binding fragment or antibody of the invention (described previously) optionally has at minimum one of the following characteristics. It detects CTLA4 levels in samples, crosses-reacts to cyno CTLA4, blocks CTLA4 binding to B7, regulates (e.g. down-regulates or regulates) CTLA4 activity or CTLA4, activates lymphocytes, and/or increases IFN-expression in lymphocytes. “in T lymphocytes.”

“In one embodiment, this invention relates to the hybridoma line LT002 deposited at China Center for Typical Culture Collection on Jun. 16, 2015, under CCTCC C201587.”

“In one embodiment, this invention relates to monoclonal antibodies produced by hybridoma cell lines LT002 and deposited at China Center for Typical Culture Collections (CCTCC), Jun. 16, 2015, under CCTCC C201587.

“The invention also covers isolated polypeptides containing the amino acid sequence of any of SEQ ID Nos. 4, 6, 8, 10, 12, 14 16, 18, 19, or 20.”

“The invention also refers to pharmaceutical compositions that contain any of the antigen binding fragments or antibodies of the invention. The invention may include any of the antigen binding fragments or antibodies of the invention. It also includes a pharmaceutically acceptable carrier/excipient. One embodiment of the invention includes any of the antigen binding fragments or antibodies of the invention and also contains a second therapeutic agent. An anti PD1 antibody, an antigen-binding fragment thereof, an anti -LAG3 antibody or antigen-binding fragment thereof, an anti TIM3 antibody or antigen-binding fragment thereof, an anti TIM3 antibody or antigen-binding fragment thereof and an anti ICOS antibody. One embodiment of the anti-PD1 antibody, or an antigen bound fragment thereof, is pembrolizumab.

“The invention also includes a conjugate containing an antigen binding fragment or antibody according to the invention, and a conjugated moiety. The conjugated moiety can be a detectable label in one embodiment. One embodiment of the conjugated moiety includes a radioisotope or fluorescent substance, an enzyme, a luminescent material, or a chromogenic substances.

“The invention also includes a kit that contains an antibody or antigen-binding fragment thereof according the invention or a conjugate according the invention and a second antigen which recognizes the antibody or antigen binding fraction thereof.” The second antibody may also include a detectable label such as a radioisotope or fluorescent substance, an enzyme, or a chromogenic material.

“The invention also includes nucleic acid molecules that encode the antigen binding fragments or antibodies of the invention or the polypeptides. The nucleic acid sequence SEQ ID NO. 3 is one embodiment of the invention. In another embodiment, it comprises the nucleic acids sequence of SEQID NO:5. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:7. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:9. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:11. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:13. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:15. Another embodiment of the invention includes the nucleic acids sequence of SEQID NO:17. The invention also includes vectors containing the nucleic acid sequence of SEQ ID NO:17 and host cells containing the nucleic acid sequence or vectors of invention.

“The invention also includes a method for producing an antigen or binding fragment. This involves: (i) cultivating a host cell with a polynucleotide that encodes the heavy chain or the light chain of any of the antigen binding pieces or antibodies of the invention under conditions conducive to expression; (ii) optionally recovering the antigen or binding fragment from the culture medium and/or host cell.”

The invention also includes a method for treating cancer in a person. This involves administering an effective amount anti-PD1 antibody or an antigen binding fragment thereof to the subject. An anti PD1 antibody, an antigen-binding fragment thereof, an anti VISTA antibody, an antigen-binding fragment thereof, an anti TIM3 antibody and an antigen-binding fragment thereof, an anti TIM3 antibody/an antigen-binding fragment thereof, an anti ICOS antibody/an antigen-binding fragment thereof or anti PDL1 antibody/an antigen-binding fragment thereof, an anti ILT4 antibody/an antigen-binding fragment thereof, an anti -CD73 antibody/an antigen-CD47 antibody/an antigen-CD47 or a fragment of the latter. One embodiment of the anti-PD1 antibodies or antigen binding pieces thereof can be selected from the following: pembrolizumab, an antigen bind fragment thereof, nivolumab, or an antigen bind fragment thereof.

The invention also includes a method for treating infection or other infectious diseases in humans. This involves administering an effective amount anti-PD1 antibody or an antigen binding fragment thereof to the subject. An anti PD1 antibody, an antigen-binding fragment thereof, an anti VISTA antibody, an antigen-binding fragment thereof, an anti TIM3 antibody and an antigen-binding fragment thereof, an anti TIM3 antibody/an antigen-binding fragment thereof, an anti ICOS antibody/an antigen-binding fragment thereof or anti PDL2 antibody; an anti ILT4 antibody; antigen-binding fragment thereof; anti ILT5 antibody; anti ILT5 or antigen-CD73 antibody; or antigen-binding fragment of the antigen-CD47 antibody; or antigen-binding fragment or antigen-binding fragment or antigen-binding fragment; or antigen-CD7 antibody; or antigen-CD47 antibody; or antigen-CD47 antibody; or antigen-binding fragment; or antigen-ILT5 or antigen-binding fragment or antigen-binding fragment or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or antigen-ILT5 or a-ILT5 or antigen-ILT5 or antigen-CD47; or if One embodiment of the anti-PD1 antibodies or antigen binding pieces thereof can be selected from the following: pembrolizumab, an antigen bind fragment thereof, nivolumab, or an antigen bind fragment thereof.

“The invention also includes a vaccine that contains an antigen binding fragment or antibody of the invention as well as an antigen.”

“The invention also includes a method of detecting the existence of a CTLA4 protein or a fragment thereof within a sample. This involves contacting the sample using an antibody or antigen binding piece of the invention, and then detecting the presence a complex between the fragment or the peptide. The detection of this complex signifies the presence CTLA4.

“The invention also includes a method for increasing the activity of an immuno cell. This involves contacting the immune cells with any of the antigen binding fragments or antibodies.”

“In one embodiment, the invention consists of a method to increase the activity of an immuno cell. This involves administering an effective amount antigen binding fragments or antibodies to the subject. This method can be used to treat cancer, an infection or infectious disease, or as a vaccine adjuvant.

“In one embodiment, an antigen or antibody binding fragment of the invention is used to prepare a medicament that can be used to increase immune cell activation, treat cancer, or treat infection or other infectious diseases.

“In one embodiment, the invention includes the use of an antigen binding fragment or antibody of the invention to make a medicament for treating cancer. This can be used for treating immune cell activation; cancer treatment; or infection or infectious disease.

“Another aspect of the invention is the use of the antigen binding fragment or antibody according to any of the embodiments to make a kit to detect the presence or amount of CTLA4 within a sample.”

“Another aspect of the invention is the use of the antigen binding fragment or antibody according to any of the embodiments or conjugates according to the invention in preparation of medicaments for the treatment or prevention of tumor, cancer, infection, or adjuvant therapy or diagnosis.”

“Another aspect of the invention is the use of the antibody/antigen binding fragment thereof according any one of the embodiments or the conjugate according the present invention in preparation of an agent that:

“Another aspect of the invention is the administration to cells of an effective amount antigen binding fragment or antibody according to any of the embodiments or conjugates according to the present inventor. The method can be selected from the following:

“Another aspect of this invention is a method for the prevention, treatment and/or adjuvant treatment and/or diagnosis a tumor or carcinoma. This involves administering to a subject an appropriate amount of the antigen binding fragment or antibody according to any of the embodiments or conjugates according to the invention.

“The antibody/antigen binding fragment of it according to any of the embodiments in the present invention for the prevention, treatment and/or adjuvant therapies and/or diagnosis a tumor/cancer.”

“The antibody/antigen binding fragment of it according to any of the embodiments in the present invention for use:

“Unless otherwise stated, technical and scientific terms used herein are the same as those who are skilled in the art. The most widely used methods in the relevant art include cell culture, molecular gene chemistry, and immunology. Below are definitions and explanations for relevant terms to help you better understand the invention.

“As used herein, when reference is made to the amino acid sequence of the CTLA4 protein (CytotoxicT-LymphocyteAntigen4), it includes the full length of the CTLA4 protein, or the extracellular fragment of CTLA4, CTLA4ECD (the portion of SEQ ID NO: 1 underlined with a wavy line), or a fragment comprising CTLA4ECD; it also includes a fusion protein of CTLA4ECD, e.g., the CTLA4ECD fragment fused to the Fc protein fragment of a mouse or human IgG (mFc or hFc) (see the description in Example 1). As those who are skilled in the art know, mutations or variations (including substitution, deletion, and/or addition) can be introduced to the CTLA4 protein’s amino acid sequence without affecting its biological function. The term “CTLA4 protein” is used in the present invention. All such sequences should be included, including the sequence underlined with an wavy line of SEQ ID No: 1, as well as the native and artificial variants. When a reference is made to the CTLA4 sequence fragment, it does not just include the sequence fragment of SEQID NO: 1, underlined with an wavy line but also the corresponding sequence fragments from its native or artificial variations.

“Sayned herein, except where otherwise stated, B7 refers only to B7-1 or B7-2. Their specific proteins sequences refers to sequences that are known to be in the art. You can refer to the sequences described in the literatures or GenBank.

“The term EC50, as used herein, refers to the concentration that causes 50% of the maximum effect.

“Antibody” is the term used herein. An immunoglobulin molecule that consists of two polypeptide chains, each with a?light? chain, is referred to as an antibody. A?heavy? and an L chain are two types of immunoglobulin molecules. (H) chain). You can classify antibody light chains as: Antibody light chains can be classified as? ???? light chain. You can classify heavy chains as??,?,?,????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????,??,??,? You can classify heavy chains as?,?,??,?,?????? or!, while the antibody type is IgM, IgD and IgG respectively. A?J? joins a light and heavy chain by joining a variable and constant regions. A region of approximately 12 or more amino acid is found in a light chain. The heavy chain also contains a?D?? A region of approximately 3 or more amino acid. Each heavy chain is composed of a heavy-chain variable region (VH), and a heavier chain constant region (CH). The heavy chain constant area consists of three domains (CH1,CH2 and CH3). Each light chain is composed of a light-chain variable region (VL), and a light-chain constant region (CL). The CL domain is the light chain constant region. The constant region of antibody mediates the binding of immunoglobulins to host tissues and factors. This includes various cells of immune system (e.g. effector cells) as well as the first component (C1q) of the classical complement system. The VH and VL regions are further subdivided into regions with high variability (referred as complementarity-determining region (CDR),) interspersed by regions known as framework regions (FR), which are more conserved. Each VH and VL is composed of 3CDRs or 4FRs. They are arranged in the following order, from the amino to the carboxy terminals: FR1, CDR1, CDR2, CDR2, CDR2, and FR3, CDR3, FR4, and FR5. Each pair of heavy and light chains has a variable region (VH) that forms an antigen binding spot. Each domain or region is assigned amino acids according to the Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health), Bethesda Md. (1987 and 1991), Chothia & Lesk (87) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342(888-883). “Antibody” is a generic term. The term?antibody? does not refer to any particular method of producing the antibody. It includes monoclonal, recombinant, and polyclonal antibody production methods. There are many types of antibodies, such as IgG (e.g. IgG1, IgG2, IgG3 subtype), IgA1, IgA2, IgD and IgE antibodies.

“Antigen binding fragment” is the term used herein. An antibody is a polypeptide that contains a fragment from a full-length antibody. This allows it to bind specifically to the antigen and compete with the full-length antibody to bind specifically to the antigen. It is also known as the?antigen binding section?. See Fundamental Immunology, Chapter. 7 (Paul W. ed. Second Edition Raven Press, N.Y. (1989),) which is incorporated by reference in its entirety herein for all purposes. Recombinant DNA techniques or chemical or enzyme cleavage can produce antigen binding fragments of antibody. In some cases, antigen binding fragments include Fab, Fab?, F(ab? ?2, Fd, Fv and dAb fragments, antigen binding fragments include Fab, Fab?, and F(ab)

“Fd fragment” is the term used herein. An antibody fragment containing the VH, CH1 and CH2 domains. The term ‘Fv fragment’? Refers to an antibody fragment that consists of the VL, VH domains of one arm of antibody. The term?dAbfragment? Refers to an antibody fragment that includes the VH domain (Ward and al. Nature 341:544-546 (1989); the term ‘Fab fragment? Refers to an antibody fragment that includes the VL, VH and CL domains. The term ‘F(ab)? )2 fragment? Refers to an antibody fragment that consists of two Fab fragments linked by disulfide bridges in the hinge region.

“In some cases the antigen binding fragment of an antibody is a single-chain antibody (e.g. scFv), in which the VL domain and VH domains pair up via a linker, which allows the production of a single-chain polypeptide chain to make a monovalent molecule (see, for example, Bird et., Science 242(4):423-426 (1988), and Huston et.l., Proc. Natl. Acad. Sci. USA 85:5879 -5883 (1988). Such scFv molecule can have the general structure: NH2?VL-Linker-VH-COOH or NH2?VH-Linker-VL-COOH. The repeated GGGGS amino acids sequence or its variants are suitable linkers in the prior art. A linker with the amino acid sequence (GGGGS4)4 may be used. However, its variants are also possible (Holliger and al. (1993)Proc. Natl. Acad. Sci. USA 90: 6444?6448 Alfthan et al. also describe other linkers that are useful in the invention. (1995) Protein Eng. 8:725-731; Choi et al. (2001) Eur. J. Immunol. 31: 94-106; Hu et al. (1996) Cancer Res. 56:3055-3061; Kipriyanov et al. (1999) J. Mol. Biol. 293:41-56 and Roovers et al. (2001) Cancer Immunol.”

“In certain cases, the antigen binding fraction of antibody is a monobody (a bivalent antibody), in which the VH domains and the VL domains are expressed on one polypeptide chain. The linker used is so short that two domains cannot pair with one another and must be paired with the complemental domain of another chain. This creates two antigen binding site (see Holliger P. and al., Proc. Natl. Acad. Sci. Sci.

“Antigen binding fragments (e.g. the above antibody fragments), can be obtained from given antibodies using conventional techniques which are well-known to those skilled in this art (e.g. recombinantDNA technique or enzymatic cleavage or chemical cleavage methods) and can be screened for specificity in much the same way as intact antibodies.

“Herein, except where otherwise indicated, includes intact antibodies as well as antigen binding fragments.

“As used herein the terms’mAb? “Molecular antibody” or “monoclonal antibodies?” An antibody or fragment of an antibody from a large number of homogenous antibody molecules. Monoclonal antibodies can be highly specific for a single epitope of the antigen. Polyclonal antibodies are more specific than monoclonal antibodies. They typically contain at least two or three antibodies that recognize different epitopes of the antigen. The hybridoma method, first described by Kohler and colleagues, is a good way to obtain monoclonal antibodies. (Nature 256:495,1975) or the recombinant-DNA technique (see U.S. Pat. No. No.

“Chimeric” is the term used herein. “Antibodies” refers to antibodies that are derived from antibodies (which can either be derived form a specific species or belong to a certain antibody class or class), while another part of the light and/or heavy chains is derived (which can also be derived (or derived) from an identical species or belonging an identical or differing antibody class or class), so long as they still have the ability to bind to the target antibody (U.S. Pat. No. Cabilly et al. ; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).”

“As used herein the term “humanized” means: “Antibodies” refers to antibodies or fragments of antibodies obtained by replacing any or all of the CDRs from a human immunoglobulin (recipient antibodies) with CDRs from a nonhuman antibody (donor antibodies). The donor antibody can be either a mouse, rat, or rabbit antibody that exhibits the desired specificity and affinity. To further enhance or optimize the performance of an antibody, it is possible to replace some of the amino acids in the recipient antibody’s framework regions (FRs), with the corresponding amino acids of the non-human antibody. For more details about humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2593-596 (1992); Clark, Immunol. Today 21: 397-402 (2000).

“Epitope” is the term used herein. Refers to the portion of an antigen that is bound specifically by an antibody or immunoglobulin. The art term “epitope” is used to refer to the part of an antigen that has been bound by an antibody or immunoglobulin. is also known as?antigenic determinaant?

An epitope, or antigenic determinant, generally consists only of the active surface group of the molecule. These include amino acids, carbohydrate compounds, or sugar side chains. They also have specific three-dimensional structural characteristics, and specific charge characteristics. An epitope can be composed of at least 3, 4, 5, 6/7, 8, 9, 10, 11, 12, 13 or 14 consecutive or inconsecutive amino acids in a far spatial conformation. It could be a ‘linear? It can be a?linear? epitope. Epitope Mapping Protocols In Methods in Molecular Biology Vol. 66, G. E. Morris, Ed. (1996). A linear epitope is a structure that contains all points of interaction between the protein (e.g., antibodies) along a primary amino acid sequence. Conformational epitopes have the inter-interacting points as they span the amino acid residues that are distinct from one another.

“Isolated anti-CTLA4 antibodies, antigen-binding pieces thereof and methods for their use are all included in the present invention. Antibody or antigen binding fragments are at least partially free from other biological molecules in the cells or cell culture where they were produced. These biological molecules can include nucleic acid, proteins, lipids and carbohydrates. A fragment of an antigen-binding antibody or other protein may be isolated from expression system components, such as biological molecules that are present in a host cell or the growth medium. The term “isolated” is generally used to refer to a cell that has been isolated from other cells. The term “isolated” does not refer to complete absence of these biological molecules, water, buffers or salts, or components of a pharmaceutical formula that include the antibodies or fragments.

“?Isolated nucleic acids molecule?” “?Isolated nucleic acid molecule” or “?isolated mononucleotide?” A DNA orRNA of genomic, molecular, or synthetic origin, which is not associated or linked to any polynucleotide. This disclosure should clarify that “a nucleic acid molecular comprising” does not include any nucleic acids. A particular sequence of nucleotides does not include intact chromosomes. Isolated nucleic acids molecules? Specific nucleic acids sequences can include, in addition, the specified sequences. Coding sequences for upto ten or more proteins, portions, or fragments thereof. Or may include operably linked regulatory sequencings that control expression.

“As used herein the term?E. What is the coli expression system? An expression system that consists of E.coli (strain), vector, and where E.coli (strain), is derived from commercial strains, such as GI698, E.2656, E.321, E.322, E.323, E.324, E.324, and E.324.

“Vector” is the term used herein. A nucleic acid carrying instrument into which a single polynucleotide may be inserted. Expression vectors are those that enable the expression of the protein encoded in the inserted polynucleotide. The vector can be introduced to a host cell through transformation, transduction, or transfection so that the gene component carried by the vector is expressed within the host cell. The art of vectors is well-known to those who are skilled in it. They include plasmid, cosmid, and artificial chromosomes, such as yeast artificial chromosomes (YAC), bacteria artificial chromosomes (BAC), or P1-derived artificial Chromosomes (PAC); bacteriophage (e.g.?). phage, M13 phage, and animal virus. Retroviruses, adenoviruses, adenoviruses, herpes viruses (e.g. herpes simplex virus), poxviruses, baculoviruses, papilloma and papova viruses (e.g. SV40) are all examples of animal viruses that can be used to create vectors. A vector may contain several components that control the expression of the gene, such as a promoter sequence and transcription initiation sequence, enhancer sequencing, selection component, reporter gene, and selector sequence. A vector may also contain a replication site.

“The term “host cell” is used herein. “Host cell” refers to any cell that can be used to introduce a vector. This includes prokaryotic and fungal cells like E. coli and Bacillus subtilis, as well as insect cells such S2 Drosophila cells or Sf9 as well as animal cells such fibroblast, CHOcells, COScells, NSOcells, HeLacells, BHKcells, HEK293cells, human cells.

“Specific binding” is the term used herein. The non-random binding reaction that occurs between two molecules, such a reaction between an antibody or its targeted antigen, is called?specific binding. An antibody that binds an antigen specifically (or an antibody specific to an antigen) in some embodiments means that the affinity (KD), of the antibody, is less than 10?5 M.

“KD” is the term used herein. The dissociation equilibrium of an antibody-antigen interaction is what’s used herein. It describes the binding affinity between the antigen and the antibody. The closer the antibody-antigen interaction is to the antigen, the greater the affinity between them. An antibody binds to the antigen when its dissociation constant (KD), is less than 10?5 M. This could be determined by surface plasmon resonance (SPR), on a BIACORE instrument, or a comparable technique (e.g. OCTET and KINEXA.

“As used herein the terms’monoclonal antibodies? “Monoclonal antibody” and “mAb?” are interchangeable terms. Both terms have the same meaning and can be interchangeably. The terms?polyclonal antibodies? are also interchangeable. The terms?polyclonal antibody? and?pub? have the same meanings. Both terms have the same meaning and can be interchangeably. The terms?polypeptide and?protein? are interchangeable. The terms?polypeptide? and?protein? have the same meanings. Both terms have the same meaning and can be interchangeably used. The present invention generally uses single-letter and three-letter abbreviations to represent amino acids. Alanine, for example, can be represented as Ala or A.

“The terms “hybridoma” and “hybridoma cell line” are used herein. Hybridoma cell line and?hybridoma? They can be interchangeable. When the term “hybridoma” is used, it also includes the subclonal and descendent cells of the hybridoma. It also includes the subclonal cells and descendent cells of hybridoma. When LT002 and LT003 are mentioned, it also includes the subclonal cells and descendent cells from the hybridoma line LT002 and LT003.

“Pharmaceutically acceptable carrier/excipient” is the term used herein. The term “pharmaceutically acceptable carrier and/or excipient” refers to a vector and/or an excipient compatible with the subject and the active part in pharmacology/physiology. These terms are well-known in the art (see Remington’s Pharmaceutical Sciences). Gennaro A. R., 19th edition. Pennsylvania: Mack Publishing Company 1995). They include, but are not limited to, pH adjusting agent and surfactant as well as adjuvant and ionic intens enhancer. The pH adjusting agent, for example, includes phosphate buffer. Surfactant can include anionic, cationic or nonionic surfactants, such as Tween 80, and ionic intensifier includes sodium chloride.

“Adjuvant” is the term used herein. “Adjuvant” is a non-specific immune enhancer that can increase or alter the immune response to an antigen. It may be delivered in combination with the antigen or before it enters the body. There are many adjuvants available, including aluminum adjuvant, such as aluminum hydroxide, Freund’s adjuvant, Corynebacterium Parvum, lipopolysaccharide and cytokine. The most widely used adjuvant in animal experiments is Freund’s, while aluminum hydroxide is used in clinical trials.

“Effective amount” is the term used herein. The effective amount is the amount that can achieve or at least partially achieve desired effects. Prophylactically effective amounts for diseases (e.g. a tumor or disease associated with CTLA4 activation or excessive binding to CTLA4) refer to an amount that is sufficient to stop, arrest or delay the development or progression of the disease. Therapeutically effective amounts for diseases refer to the amount that can cure the disease or at least partially stop its complications for the patient. This is something that can be done by those who are skilled in the art. A therapeutically effective amount will, for example, depend on the severity and condition of the disease, the general state of the immune system, the general health of the patient, and other factors such as age, body mass, weight, administration mode, and other therapies that are being administered simultaneously.

“As used in this document, including the appended Claims, the singular forms words such as ‘a,? ?an,? If the context is clear,?an? and?the? should be used together.

“?Administration? “?Administration” refers to any contact of an exogenous therapeutic, diagnostic, or pharmaceutical agent or composition with an animal, human subject, cell tissue, organ or animal.

“?Treat? “?Treat? The act of administering a therapeutic agent (e.g., a composition containing one of the antigen-binding fragments or antibodies of the invention) to an individual or subject suffering from one or more of the diseases or suspected of developing one, or both, of these symptoms. The agent is usually administered in a dose that is effective in relieving one or more symptoms of a disease in the subject or population. This may be done by either inducing or inhibiting the progression of the symptom(s). The effectiveness of any therapeutic agent to relieve a particular disease symptom will vary depending on factors like the age and weight of patients and the drug’s ability to induce a desired response. Any clinical measurement that is used by doctors or other skilled healthcare providers can help determine if a disease symptom has been relieved.

“Antibodies of the Invention.”

Monoclonal antibodies (MAB), which are therapeutic antibodies, have shown remarkable efficacy in treating a variety of diseases. The traditional way to obtain therapeutic antibodies is to immunize an animal with an antigen and then obtain antibodies against that antigen from the animal. Optionally, affinity maturation can be used to improve an antibody with low affinity to the antigen. This method can be labor-intensive and time-consuming. It also fails to pinpoint a specific epitope of the antigen.

“Antigen binding depends on the variable regions in the light and heavy chains; the variable regions of each chain comprise three hypervariable areas, also known as complementarity determining regions (CDR). The heavy chain (H), which includes HCDR1, HCD2 and HCDR3, while the light chain (L), comprises LCDR1, LCDR2 or LCDR3. For definitions, refer to Kabat et al. Sequences Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 93-3282, Bethesda MD

“The invention is in part related to anti-CTLA4 antibody sequences. The invention’s antibodies can specifically bind CTLA4. They are able to block CLTA4 from B7 binding, relieve CTLA4-induced immunosuppression and activate T lymphocytes.

“In particular, this invention refers to the mouse antibody 4G10 described herein. It comprises the heavy chain variable area (VH) and the light-chain variable region (VL), both of which are SEQ ID No:4; and humanized variants of this antibody.

“In certain embodiments, the humanized VH-sequences of the 4G10 antibody can include any of the following VH areas:

“In certain embodiments, the humanized VH sequence for the 4G10 antibody may include the following CDR areas (determined according to Kabat:”

“In alternate embodiments, the humanized VH sequence for the 4G10 antibody may include the following CDR areas (determined using VBASE2 database analysis:”

“In certain embodiments, the 4G10 antibody’s humanized VL sequences can include any of the following VL areas:

“In certain embodiments, the humanized sequence of the 4G10 antibody can include the following CDR areas (which were determined according to Kabat:

“In alternative embodiments, a humanized VL sequence for the 4G10 antibody may include the following CDR areas (which CDR areas were determined using VBASE database analysis).

“Any of these humanized VH regions can be paired with any one of the VL regions. Preferential embodiments include an antibody that includes the VH region in SEQ ID No: 19 and VL region in SEQ ID No: 20. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 8. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 12. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 16 and 14. Preferential embodiments include an antibody that includes the VH region and VL regions of SEQID NO: 18 and SEQID NO: 14.

“Antibody Expression”

“The invention’s antibodies and fragments can be made by any skilled person in the art. The invention’s antibodies and antibody fragments can be made in any cell. Eukaryotic and prokaryotic cells can be used as hosts to express the antibodies or fragments of immunoglobulin chains. This includes mammalian cells. These cells include, among others, Chinese hamster eggs (CHO), NSO, SP2 and HeLa cells. They also include baby hamster kidney (BHK), monkey kidney (COS) cells and human hepatocellular carcinoma (e.g. Hep G2) cells. Mammalian host cell types include mammalian human, mouse and rat cells, as well as bovine, horse, and hamster cells. Selection of cell lines that express high levels of expression is a way to identify cell lines of preference. Sf9 cells, plant cell lines, and amphibian cell lines are all possible cell types. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia ptjperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp. any Saccharomyces sp. Hansenula polymorpha any Kluyveromyces sp. Candida albicans any Aspergillus sp. Trichoderma reesei Chrysosporium luckynowense, Fusarium sp. Yarrowia crassa Recombinant expression vectors that encode the antigen-binding or heavy chain portion or fragment, and the light chain or antigen binding fragment thereof are introduced to mammalian host cell. The antibodies are created by cultivating the host cells for sufficient time to permit the expression or secretion of the antigen or fragment in the host cell or into the culture medium in order to produce them.

“Antibody Purification”

Standard protein purification methods can be used to recover antibodies and antigen binding fragments and immunoglobulin chain from culture media. There are many techniques that can be used to increase the expression of antigen-binding and antibody fragments and immunoglobulin chain of the invention or other moieties from cell lines. The GS system, which is a common method of increasing expression in certain circumstances, is an example. European Patent Nos. are discussed in part or whole. 0 216 846; 0 256 5055, 0 323 997, and European Patent Application No. 89303964.4. In one embodiment of the invention, mammalian host cells (e.g. CHO) lack a glutamine synetase genes and are grown in medium without glutamine. However, the polynucleotide encoding immunoglobulin chains contains a glutamine synthetase genetic which is complementary to the gene’s absence in the host cell.

The present invention provides methods for purifying an antigen-binding or antibody fragment of the invention. These methods include introducing the sample containing the antibody or fragment to a purification media (e.g. anion exchange medium or cation exchange medium), and then either collecting the purified antibody or fragment or discarding the flow through fraction. Finally, eluting the bound antibody from the medium and collecting the liquid eluate. The medium is contained in the column to which the sample is being applied. The purification process is performed according to an embodiment of the invention. For example, the medium is placed in a column to which the sample is applied.

“Antibody Engineering”

“In some embodiments, antigen-binding fragments and antibodies of the invention can be modified to enhance their properties by including modifications to the framework and/or CDRs. These engineered modifications can be made using molecular modeling. To understand the structure of the antibody, a molecular model can be built for the variable region in the non-human parental antibody sequence. This can then be used to identify regions that could interact with the antigen. Conventional CDRs are built on the alignment of immunoglobulins sequences and identification of variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al. ; National Institutes of Health Bethesda (Md. ; 5th ed. NIH Publ. No. No. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and her coworkers examined the conformations of loops in crystal structures for antibodies and suggested hypervariable loops. Chothia, et al., (1987) J. Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are differences between the?CDRs? regions. These include?hypervariable loops’ and?CDRs?. Later studies (Raghunathan et al, (2012) J Mol Recog. 25, 3, 103-113) Analyzing several antibody?antigen complexes, we found that antigen binding areas in antibodies don’t always conform to the?CDR. Antigen binding regions in antibodies do not always conform to the?CDR?. loops. To guide the selection of potential binding regions to the antigen, the molecular model of the non-human antibody variable region can be used. The model-based potential binding regions of antigen differ from conventional?CDRs or ‘hyper variable? in practice. loops. For molecular modeling, commercial scientific software like MOE (Chemical Computing Group), can be used. The best matches between the human frameworks and the non-human sequence can be used to select the human frameworks. The VJ regions of the human germlines that are part of FR4 (framework 4 in VH) are compared to the non-human counterpart. For FR4 in VL, JKappa and JLambda are compared to the corresponding region of the non-human germline sequences. After identifying suitable human frameworks, the CDRs can be grafted into those frameworks. Some residues at the VL-VH interface may be retained in some cases. For identifying potential CDR conformations that could alter and binding to antigen, Molecular models are also useful. These residues may be retained in their non-human (parental sequence) form. You can also use molecular models to identify solvent-exposed amino acids that could cause undesirable effects like glycosylation or deamidation. These potential problems can be eliminated or minimized by using developability filters.

“Another method of modifying the framework is to modify one or more residues in the framework region or within one or several CDR regions to remove T cell epitopes and reduce the antibody’s immunogenicity. This is sometimes called “deimmunization”. This approach is also known as?deimmunization?. No. 7,125,689.”

“In certain embodiments, it may be possible to alter some amino acids that have exposed side-chains to another residue to increase the chemical stability of final antibodies. This will avoid isomerization or deamidation. Asparagine can be deamidated on NG or DG. NG, NS and NA sequences. This creates an isoaspartic acids residue which introduces a kink in the polypeptide chain and decreases its stability. Isomerization can occur at DG or DS sequences. The antibodies described in the present disclosure may not contain asparagine or deamidation isomerism sites.

“For example, an Asparagine (Asn), residue can be changed to Gln, or Ala, to decrease the possibility of formation isoaspartate at Asn-Gly sequences. This is especially true for a CDR. Similar problems could occur in an Asp-Gly sequence. Reissner & Aswad, 2003 Cell. Mol. Life Sci. 60:1281. The formation of isoaspartate may cause the binding of an antibody against its target antigen to be disrupted or even broken. Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. One embodiment changes the asparagine to glutamine (Gln). To reduce the risk of deamidation (which occurs more frequently when small amino acids are near asparagine and glutamine), it may be possible to change an amino acid that is adjacent to an asparagine residue (Asn). See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. 662:261. Id. Id.

“Exemplary Stabilizing CDR Type Variants”

“CDR Residue Managing Variant Sequence”

“Antibody Engineering of the Fe Region.”

“Antibodies (e.g. humanized antibodies) can be modified to alter the Fc region. This is typically done to alter one or more properties, such as serum half life, complement fixation and Fc receptor binding and/or effector functions (e.g. antigen-dependent cell cytotoxicity). The antigen-binding pieces and antibodies described herein can also be chemically modified. For example, one or several chemical moieties can attach to the antibody, or modified to alter its glycosylation. This can alter any or all of the fragment’s properties.

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