US20210095026A1 - Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) - Google Patents
Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) Download PDFInfo
- Publication number
- US20210095026A1 US20210095026A1 US17/074,269 US202017074269A US2021095026A1 US 20210095026 A1 US20210095026 A1 US 20210095026A1 US 202017074269 A US202017074269 A US 202017074269A US 2021095026 A1 US2021095026 A1 US 2021095026A1
- Authority
- US
- United States
- Prior art keywords
- lag
- antibody
- cancer
- seq
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 title claims abstract description 229
- 102100020862 Lymphocyte activation gene 3 protein Human genes 0.000 title claims abstract description 203
- 238000000034 method Methods 0.000 claims abstract description 68
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 claims abstract description 47
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 claims abstract description 47
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 claims abstract description 40
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 claims abstract description 40
- 230000027455 binding Effects 0.000 claims abstract description 40
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 36
- 201000011510 cancer Diseases 0.000 claims abstract description 15
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 claims description 9
- 208000029742 colonic neoplasm Diseases 0.000 claims description 8
- 201000001441 melanoma Diseases 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 5
- 201000005202 lung cancer Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 206010006187 Breast cancer Diseases 0.000 claims description 3
- 208000026310 Breast neoplasm Diseases 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 3
- 208000022072 Gallbladder Neoplasms Diseases 0.000 claims description 3
- 206010023825 Laryngeal cancer Diseases 0.000 claims description 3
- 208000002030 Merkel cell carcinoma Diseases 0.000 claims description 3
- 206010029266 Neuroendocrine carcinoma of the skin Diseases 0.000 claims description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 3
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 claims description 3
- 206010061934 Salivary gland cancer Diseases 0.000 claims description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 3
- 201000010881 cervical cancer Diseases 0.000 claims description 3
- 208000017763 cutaneous neuroendocrine carcinoma Diseases 0.000 claims description 3
- 201000010175 gallbladder cancer Diseases 0.000 claims description 3
- 206010017758 gastric cancer Diseases 0.000 claims description 3
- 206010023841 laryngeal neoplasm Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 3
- 201000002528 pancreatic cancer Diseases 0.000 claims description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 3
- 201000011549 stomach cancer Diseases 0.000 claims description 3
- 201000002510 thyroid cancer Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 121
- 229920001184 polypeptide Polymers 0.000 abstract description 118
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 118
- 239000011230 binding agent Substances 0.000 abstract description 75
- 108090000623 proteins and genes Proteins 0.000 abstract description 39
- 239000013598 vector Substances 0.000 abstract description 33
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 27
- 102000004169 proteins and genes Human genes 0.000 abstract description 27
- 239000000203 mixture Substances 0.000 abstract description 22
- 201000010099 disease Diseases 0.000 abstract description 15
- 208000035475 disorder Diseases 0.000 abstract description 12
- 239000003795 chemical substances by application Substances 0.000 abstract description 11
- 230000005764 inhibitory process Effects 0.000 abstract description 10
- 208000035473 Communicable disease Diseases 0.000 abstract description 8
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 99
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 59
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 55
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 44
- 235000001014 amino acid Nutrition 0.000 description 42
- 125000003275 alpha amino acid group Chemical group 0.000 description 40
- 229940024606 amino acid Drugs 0.000 description 38
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 37
- 150000001413 amino acids Chemical class 0.000 description 35
- 150000007523 nucleic acids Chemical group 0.000 description 34
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 31
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 31
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 30
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 29
- 235000009582 asparagine Nutrition 0.000 description 29
- 229960001230 asparagine Drugs 0.000 description 29
- 230000014509 gene expression Effects 0.000 description 29
- 239000000427 antigen Substances 0.000 description 28
- 108091007433 antigens Proteins 0.000 description 28
- 102000036639 antigens Human genes 0.000 description 28
- 230000000694 effects Effects 0.000 description 28
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 27
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 27
- 241001465754 Metazoa Species 0.000 description 27
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 23
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 21
- 210000001744 T-lymphocyte Anatomy 0.000 description 21
- 239000012634 fragment Substances 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 21
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 18
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 230000006870 function Effects 0.000 description 18
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 17
- 238000003556 assay Methods 0.000 description 17
- 239000004472 Lysine Substances 0.000 description 16
- 235000003704 aspartic acid Nutrition 0.000 description 16
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 16
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 16
- 229960000310 isoleucine Drugs 0.000 description 16
- 235000018977 lysine Nutrition 0.000 description 16
- 239000004471 Glycine Substances 0.000 description 15
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 15
- 235000004279 alanine Nutrition 0.000 description 15
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 14
- 239000012636 effector Substances 0.000 description 14
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 14
- 235000004554 glutamine Nutrition 0.000 description 14
- 108060003951 Immunoglobulin Proteins 0.000 description 13
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 13
- 241000124008 Mammalia Species 0.000 description 13
- 102000018358 immunoglobulin Human genes 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 12
- 239000004475 Arginine Substances 0.000 description 11
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 11
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 11
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 11
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 11
- 235000009697 arginine Nutrition 0.000 description 11
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 11
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 10
- 239000004473 Threonine Substances 0.000 description 10
- 210000004408 hybridoma Anatomy 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 230000004071 biological effect Effects 0.000 description 9
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 8
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 8
- 235000013922 glutamic acid Nutrition 0.000 description 8
- 239000004220 glutamic acid Substances 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 8
- 239000004474 valine Substances 0.000 description 8
- 102000000588 Interleukin-2 Human genes 0.000 description 7
- 108010002350 Interleukin-2 Proteins 0.000 description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 239000005557 antagonist Substances 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 229930182817 methionine Natural products 0.000 description 7
- 210000003289 regulatory T cell Anatomy 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- ZDILXFDENZVOTL-BPNCWPANSA-N Ala-Val-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDILXFDENZVOTL-BPNCWPANSA-N 0.000 description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 125000001931 aliphatic group Chemical group 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000006386 neutralization reaction Methods 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 5
- 230000006044 T cell activation Effects 0.000 description 5
- -1 aromatic amino acid Chemical class 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000009871 nonspecific binding Effects 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 230000004614 tumor growth Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 4
- SWWCDAGDQHTKIE-RHYQMDGZSA-N Lys-Arg-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWWCDAGDQHTKIE-RHYQMDGZSA-N 0.000 description 4
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 210000004443 dendritic cell Anatomy 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 4
- 108010037850 glycylvaline Proteins 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- IETUUAHKCHOQHP-KZVJFYERSA-N Ala-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)[C@@H](C)O)C(O)=O IETUUAHKCHOQHP-KZVJFYERSA-N 0.000 description 3
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 108010031111 EBV-encoded nuclear antigen 1 Proteins 0.000 description 3
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 3
- WKJKBELXHCTHIJ-WPRPVWTQSA-N Gly-Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N WKJKBELXHCTHIJ-WPRPVWTQSA-N 0.000 description 3
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 3
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 3
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 3
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 3
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 3
- PRSBSVAVOQOAMI-BJDJZHNGSA-N Lys-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN PRSBSVAVOQOAMI-BJDJZHNGSA-N 0.000 description 3
- 102000043131 MHC class II family Human genes 0.000 description 3
- 108091054438 MHC class II family Proteins 0.000 description 3
- 108010079364 N-glycylalanine Proteins 0.000 description 3
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 3
- 241000725643 Respiratory syncytial virus Species 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 3
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 3
- WGDYNRCOQRERLZ-KKUMJFAQSA-N Ser-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N WGDYNRCOQRERLZ-KKUMJFAQSA-N 0.000 description 3
- 102100022433 Single-stranded DNA cytosine deaminase Human genes 0.000 description 3
- 101710143275 Single-stranded DNA cytosine deaminase Proteins 0.000 description 3
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 3
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- OVLIFGQSBSNGHY-KKHAAJSZSA-N Val-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N)O OVLIFGQSBSNGHY-KKHAAJSZSA-N 0.000 description 3
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 3
- 230000009824 affinity maturation Effects 0.000 description 3
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 3
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 201000010897 colon adenocarcinoma Diseases 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- 230000013632 homeostatic process Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 210000001236 prokaryotic cell Anatomy 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108010038745 tryptophylglycine Proteins 0.000 description 3
- 108010025432 tyrosyl-alanyl-phenylalanyl-glycine Proteins 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- CWPDVLMKCHLLPS-JVPBZIDWSA-N 2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]-3-phenylpropanoyl]amino]acetic acid Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=C(O)C=C1 CWPDVLMKCHLLPS-JVPBZIDWSA-N 0.000 description 2
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 2
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 2
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 2
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 2
- PBSOQGZLPFVXPU-YUMQZZPRSA-N Arg-Glu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PBSOQGZLPFVXPU-YUMQZZPRSA-N 0.000 description 2
- AOHKLEBWKMKITA-IHRRRGAJSA-N Arg-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N AOHKLEBWKMKITA-IHRRRGAJSA-N 0.000 description 2
- QLSRIZIDQXDQHK-RCWTZXSCSA-N Arg-Val-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QLSRIZIDQXDQHK-RCWTZXSCSA-N 0.000 description 2
- AITGTTNYKAWKDR-CIUDSAMLSA-N Asn-His-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O AITGTTNYKAWKDR-CIUDSAMLSA-N 0.000 description 2
- GMUOCGCDOYYWPD-FXQIFTODSA-N Asn-Pro-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O GMUOCGCDOYYWPD-FXQIFTODSA-N 0.000 description 2
- BSWHERGFUNMWGS-UHFFFAOYSA-N Asp-Ile Chemical compound CCC(C)C(C(O)=O)NC(=O)C(N)CC(O)=O BSWHERGFUNMWGS-UHFFFAOYSA-N 0.000 description 2
- USNJAPJZSGTTPX-XVSYOHENSA-N Asp-Phe-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O USNJAPJZSGTTPX-XVSYOHENSA-N 0.000 description 2
- DWBZEJHQQIURML-IMJSIDKUSA-N Asp-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O DWBZEJHQQIURML-IMJSIDKUSA-N 0.000 description 2
- XWKPSMRPIKKDDU-RCOVLWMOSA-N Asp-Val-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O XWKPSMRPIKKDDU-RCOVLWMOSA-N 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 2
- GHAXJVNBAKGWEJ-AVGNSLFASA-N Gln-Ser-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GHAXJVNBAKGWEJ-AVGNSLFASA-N 0.000 description 2
- VLOLPWWCNKWRNB-LOKLDPHHSA-N Gln-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VLOLPWWCNKWRNB-LOKLDPHHSA-N 0.000 description 2
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 2
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 2
- SBCYJMOOHUDWDA-NUMRIWBASA-N Glu-Asp-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SBCYJMOOHUDWDA-NUMRIWBASA-N 0.000 description 2
- YQPFCZVKMUVZIN-AUTRQRHGSA-N Glu-Val-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O YQPFCZVKMUVZIN-AUTRQRHGSA-N 0.000 description 2
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 2
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 2
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 2
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 2
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 2
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 2
- WTUSRDZLLWGYAT-KCTSRDHCSA-N Gly-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN WTUSRDZLLWGYAT-KCTSRDHCSA-N 0.000 description 2
- JYGYNWYVKXENNE-OALUTQOASA-N Gly-Tyr-Trp Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JYGYNWYVKXENNE-OALUTQOASA-N 0.000 description 2
- NGBGZCUWFVVJKC-IRXDYDNUSA-N Gly-Tyr-Tyr Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NGBGZCUWFVVJKC-IRXDYDNUSA-N 0.000 description 2
- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- GTSAALPQZASLPW-KJYZGMDISA-N Ile-His-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N GTSAALPQZASLPW-KJYZGMDISA-N 0.000 description 2
- FBGXMKUWQFPHFB-JBDRJPRFSA-N Ile-Ser-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N FBGXMKUWQFPHFB-JBDRJPRFSA-N 0.000 description 2
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 2
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 2
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- UCOCBWDBHCUPQP-DCAQKATOSA-N Leu-Arg-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O UCOCBWDBHCUPQP-DCAQKATOSA-N 0.000 description 2
- CQGSYZCULZMEDE-SRVKXCTJSA-N Leu-Gln-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O CQGSYZCULZMEDE-SRVKXCTJSA-N 0.000 description 2
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 2
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 2
- ZFNLIDNJUWNIJL-WDCWCFNPSA-N Leu-Glu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZFNLIDNJUWNIJL-WDCWCFNPSA-N 0.000 description 2
- FEHQLKKBVJHSEC-SZMVWBNQSA-N Leu-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FEHQLKKBVJHSEC-SZMVWBNQSA-N 0.000 description 2
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 2
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 2
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 2
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 2
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 2
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 2
- LMDVGHQPPPLYAR-IHRRRGAJSA-N Leu-Val-His Chemical compound N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O LMDVGHQPPPLYAR-IHRRRGAJSA-N 0.000 description 2
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- XDMCWZFLLGVIID-SXPRBRBTSA-N O-(3-O-D-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine Chemical compound CC(=O)N[C@H]1[C@H](OC[C@H]([NH3+])C([O-])=O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 XDMCWZFLLGVIID-SXPRBRBTSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- YYKZDTVQHTUKDW-RYUDHWBXSA-N Phe-Gly-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N YYKZDTVQHTUKDW-RYUDHWBXSA-N 0.000 description 2
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 2
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 2
- FNGOXVQBBCMFKV-CIUDSAMLSA-N Pro-Ser-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O FNGOXVQBBCMFKV-CIUDSAMLSA-N 0.000 description 2
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- QGMLKFGTGXWAHF-IHRRRGAJSA-N Ser-Arg-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QGMLKFGTGXWAHF-IHRRRGAJSA-N 0.000 description 2
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 2
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 2
- FMDHKPRACUXATF-ACZMJKKPSA-N Ser-Gln-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O FMDHKPRACUXATF-ACZMJKKPSA-N 0.000 description 2
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 2
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 2
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 2
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 2
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 2
- UKKROEYWYIHWBD-ZKWXMUAHSA-N Ser-Val-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UKKROEYWYIHWBD-ZKWXMUAHSA-N 0.000 description 2
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000006052 T cell proliferation Effects 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- JVTHIXKSVYEWNI-JRQIVUDYSA-N Thr-Asn-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JVTHIXKSVYEWNI-JRQIVUDYSA-N 0.000 description 2
- KRPKYGOFYUNIGM-XVSYOHENSA-N Thr-Asp-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O KRPKYGOFYUNIGM-XVSYOHENSA-N 0.000 description 2
- UTCFSBBXPWKLTG-XKBZYTNZSA-N Thr-Cys-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O UTCFSBBXPWKLTG-XKBZYTNZSA-N 0.000 description 2
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 2
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 2
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 2
- JAWUQFCGNVEDRN-MEYUZBJRSA-N Thr-Tyr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O JAWUQFCGNVEDRN-MEYUZBJRSA-N 0.000 description 2
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 2
- FEZASNVQLJQBHW-CABZTGNLSA-N Trp-Gly-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O)=CNC2=C1 FEZASNVQLJQBHW-CABZTGNLSA-N 0.000 description 2
- TZXFLDNBYYGLKA-BZSNNMDCSA-N Tyr-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 TZXFLDNBYYGLKA-BZSNNMDCSA-N 0.000 description 2
- QOIKZODVIPOPDD-AVGNSLFASA-N Tyr-Cys-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QOIKZODVIPOPDD-AVGNSLFASA-N 0.000 description 2
- UMXSDHPSMROQRB-YJRXYDGGSA-N Tyr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O UMXSDHPSMROQRB-YJRXYDGGSA-N 0.000 description 2
- AKLNEFNQWLHIGY-QWRGUYRKSA-N Tyr-Gly-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N)O AKLNEFNQWLHIGY-QWRGUYRKSA-N 0.000 description 2
- 108010064997 VPY tripeptide Proteins 0.000 description 2
- QPPZEDOTPZOSEC-RCWTZXSCSA-N Val-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N)O QPPZEDOTPZOSEC-RCWTZXSCSA-N 0.000 description 2
- ZXYPHBKIZLAQTL-QXEWZRGKSA-N Val-Pro-Asp Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N ZXYPHBKIZLAQTL-QXEWZRGKSA-N 0.000 description 2
- QWCZXKIFPWPQHR-JYJNAYRXSA-N Val-Pro-Tyr Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QWCZXKIFPWPQHR-JYJNAYRXSA-N 0.000 description 2
- CEKSLIVSNNGOKH-KZVJFYERSA-N Val-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](C(C)C)N)O CEKSLIVSNNGOKH-KZVJFYERSA-N 0.000 description 2
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 2
- GUIYPEKUEMQBIK-JSGCOSHPSA-N Val-Tyr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(O)=O GUIYPEKUEMQBIK-JSGCOSHPSA-N 0.000 description 2
- PGBMPFKFKXYROZ-UFYCRDLUSA-N Val-Tyr-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N PGBMPFKFKXYROZ-UFYCRDLUSA-N 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 108010081404 acein-2 Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000000975 co-precipitation Methods 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 108010018006 histidylserine Proteins 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 210000002602 induced regulatory T cell Anatomy 0.000 description 2
- 230000004073 interleukin-2 production Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108010031719 prolyl-serine Proteins 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 108010090894 prolylleucine Proteins 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002849 thermal shift Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- IEJPPSMHUUQABK-UHFFFAOYSA-N 2,4-diphenyl-4h-1,3-oxazol-5-one Chemical compound O=C1OC(C=2C=CC=CC=2)=NC1C1=CC=CC=C1 IEJPPSMHUUQABK-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- LQJAALCCPOTJGB-YUMQZZPRSA-N Arg-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 description 1
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 1
- LGCVSPFCFXWUEY-IHPCNDPISA-N Asn-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N LGCVSPFCFXWUEY-IHPCNDPISA-N 0.000 description 1
- DATSKXOXPUAOLK-KKUMJFAQSA-N Asn-Tyr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DATSKXOXPUAOLK-KKUMJFAQSA-N 0.000 description 1
- KLYPOCBLKMPBIQ-GHCJXIJMSA-N Asp-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N KLYPOCBLKMPBIQ-GHCJXIJMSA-N 0.000 description 1
- BRRPVTUFESPTCP-ACZMJKKPSA-N Asp-Ser-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O BRRPVTUFESPTCP-ACZMJKKPSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 241000193764 Brevibacillus brevis Species 0.000 description 1
- 101100352418 Caenorhabditis elegans plp-1 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- DRXOWZZHCSBUOI-YJRXYDGGSA-N Cys-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CS)N)O DRXOWZZHCSBUOI-YJRXYDGGSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 241000725619 Dengue virus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- UPEZCKBFRMILAV-JNEQICEOSA-N Ecdysone Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@H]([C@@H](O)CCC(O)(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 UPEZCKBFRMILAV-JNEQICEOSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- XZLLTYBONVKGLO-SDDRHHMPSA-N Gln-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XZLLTYBONVKGLO-SDDRHHMPSA-N 0.000 description 1
- SYZZMPFLOLSMHL-XHNCKOQMSA-N Gln-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)C(=O)O SYZZMPFLOLSMHL-XHNCKOQMSA-N 0.000 description 1
- BYKZWDGMJLNFJY-XKBZYTNZSA-N Gln-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)O BYKZWDGMJLNFJY-XKBZYTNZSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- TUTIHHSZKFBMHM-WHFBIAKZSA-N Glu-Asn Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O TUTIHHSZKFBMHM-WHFBIAKZSA-N 0.000 description 1
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-N 0.000 description 1
- SITLTJHOQZFJGG-XPUUQOCRSA-N Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O SITLTJHOQZFJGG-XPUUQOCRSA-N 0.000 description 1
- IEFJWDNGDZAYNZ-BYPYZUCNSA-N Gly-Glu Chemical compound NCC(=O)N[C@H](C(O)=O)CCC(O)=O IEFJWDNGDZAYNZ-BYPYZUCNSA-N 0.000 description 1
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 1
- VBOBNHSVQKKTOT-YUMQZZPRSA-N Gly-Lys-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O VBOBNHSVQKKTOT-YUMQZZPRSA-N 0.000 description 1
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- AJHCSUXXECOXOY-NSHDSACASA-N Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-NSHDSACASA-N 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- FOCSWPCHUDVNLP-PMVMPFDFSA-N His-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CC4=CN=CN4)N FOCSWPCHUDVNLP-PMVMPFDFSA-N 0.000 description 1
- ZNTSGDNUITWTRA-WDSOQIARSA-N His-Trp-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O ZNTSGDNUITWTRA-WDSOQIARSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101001008255 Homo sapiens Immunoglobulin kappa variable 1D-8 Proteins 0.000 description 1
- 101001047628 Homo sapiens Immunoglobulin kappa variable 2-29 Proteins 0.000 description 1
- 101001008321 Homo sapiens Immunoglobulin kappa variable 2D-26 Proteins 0.000 description 1
- 101001047619 Homo sapiens Immunoglobulin kappa variable 3-20 Proteins 0.000 description 1
- 101001008263 Homo sapiens Immunoglobulin kappa variable 3D-15 Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 238000012450 HuMAb Mouse Methods 0.000 description 1
- 102100022949 Immunoglobulin kappa variable 2-29 Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- 102000017578 LAG3 Human genes 0.000 description 1
- USTCFDAQCLDPBD-XIRDDKMYSA-N Leu-Asn-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N USTCFDAQCLDPBD-XIRDDKMYSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- FQZPTCNSNPWHLJ-AVGNSLFASA-N Leu-Gln-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O FQZPTCNSNPWHLJ-AVGNSLFASA-N 0.000 description 1
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 1
- FKQPWMZLIIATBA-AJNGGQMLSA-N Leu-Lys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FKQPWMZLIIATBA-AJNGGQMLSA-N 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- SJDQOYTYNGZZJX-SRVKXCTJSA-N Met-Glu-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SJDQOYTYNGZZJX-SRVKXCTJSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101001137986 Mus musculus Lymphocyte activation gene 3 protein Proteins 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 102000009890 Osteonectin Human genes 0.000 description 1
- 108010077077 Osteonectin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- JMVQDLDPDBXAAX-YUMQZZPRSA-N Pro-Gly-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 JMVQDLDPDBXAAX-YUMQZZPRSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- RMODQFBNDDENCP-IHRRRGAJSA-N Pro-Lys-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O RMODQFBNDDENCP-IHRRRGAJSA-N 0.000 description 1
- RWCOTTLHDJWHRS-YUMQZZPRSA-N Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RWCOTTLHDJWHRS-YUMQZZPRSA-N 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 241000293825 Rhinosporidium Species 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- ICHZYBVODUVUKN-SRVKXCTJSA-N Ser-Asn-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ICHZYBVODUVUKN-SRVKXCTJSA-N 0.000 description 1
- NFDYGNFETJVMSE-BQBZGAKWSA-N Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CO NFDYGNFETJVMSE-BQBZGAKWSA-N 0.000 description 1
- WBAXJMCUFIXCNI-WDSKDSINSA-N Ser-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WBAXJMCUFIXCNI-WDSKDSINSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- LDEBVRIURYMKQS-WISUUJSJSA-N Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO LDEBVRIURYMKQS-WISUUJSJSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 230000006043 T cell recruitment Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- YOOAQCZYZHGUAZ-KATARQTJSA-N Thr-Leu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YOOAQCZYZHGUAZ-KATARQTJSA-N 0.000 description 1
- SVGAWGVHFIYAEE-JSGCOSHPSA-N Trp-Gly-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 SVGAWGVHFIYAEE-JSGCOSHPSA-N 0.000 description 1
- GQHAIUPYZPTADF-FDARSICLSA-N Trp-Ile-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 GQHAIUPYZPTADF-FDARSICLSA-N 0.000 description 1
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 1
- NUQZCPSZHGIYTA-HKUYNNGSSA-N Tyr-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N NUQZCPSZHGIYTA-HKUYNNGSSA-N 0.000 description 1
- 101150117115 V gene Proteins 0.000 description 1
- VCAWFLIWYNMHQP-UKJIMTQDSA-N Val-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N VCAWFLIWYNMHQP-UKJIMTQDSA-N 0.000 description 1
- OWFGFHQMSBTKLX-UFYCRDLUSA-N Val-Tyr-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N OWFGFHQMSBTKLX-UFYCRDLUSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 0.000 description 1
- 229940123627 Viral replication inhibitor Drugs 0.000 description 1
- 244000195452 Wasabia japonica Species 0.000 description 1
- 235000000760 Wasabia japonica Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- UPEZCKBFRMILAV-UHFFFAOYSA-N alpha-Ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C(O)CCC(C)(C)O)C)CCC33O)C)C3=CC(=O)C21 UPEZCKBFRMILAV-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- OIRDTQYFTABQOQ-UHFFFAOYSA-N ara-adenosine Natural products Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O OIRDTQYFTABQOQ-UHFFFAOYSA-N 0.000 description 1
- 101150035354 araA gene Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940022399 cancer vaccine Drugs 0.000 description 1
- 238000009566 cancer vaccine Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010205 computational analysis Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 210000001840 diploid cell Anatomy 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- UPEZCKBFRMILAV-JMZLNJERSA-N ecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@H]([C@H](O)CCC(C)(C)O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 UPEZCKBFRMILAV-JMZLNJERSA-N 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000012308 immunohistochemistry method Methods 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000007688 immunotoxicity Effects 0.000 description 1
- 231100000386 immunotoxicity Toxicity 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 229940028885 interleukin-4 Drugs 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000000670 ligand binding assay Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000010874 maintenance of protein location Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 108700039855 mouse a Proteins 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229940127084 other anti-cancer agent Drugs 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 210000001586 pre-b-lymphocyte Anatomy 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000001948 pro-b lymphocyte Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 238000003567 signal transduction assay Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- JOPDZQBPOWAEHC-UHFFFAOYSA-H tristrontium;diphosphate Chemical compound [Sr+2].[Sr+2].[Sr+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O JOPDZQBPOWAEHC-UHFFFAOYSA-H 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 229940126580 vector vaccine Drugs 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/524—CH2 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/71—Decreased effector function due to an Fc-modification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- Lymphocyte Activation Gene-3 which is also known as CD223, is a member of the immunoglobulin supergene family and is structurally and genetically related to CD4. LAG-3 is expressed on T-cells, B cells, natural killer (NK) cells and plasmacytoid dendritic cells (ADCs). Like CD4, LAG-3 has been demonstrated to interact with MHC Class 11 molecules (Baixeras et al., J. Exp. Med., 176: 327-337 (1992)), but binds at a distinct site (Huard et al., Proc. Natl. Acad. Sci. USA, 94(11): 5744-5749 (1997)).
- LAG-3 immunoglobulin fusion protein sLAG-3Ig directly and specifically binds via LAG-3 to MHC class II on the cell surface (Huard et al., Eur. J. Immunol., 26:1180-1186 (1996)).
- LAG-3 is upregulated following T-cell activation, and modulates T-cell function as well as T-cell homeostasis (Sierra et al., Expert Opin. Ther. Targets, 15(1):91-101 (2011)).
- the LAG-3/MHC class 11 interaction may play a role in down-regulating antigen-dependent stimulation of CD4+ T lymphocytes, as demonstrated in in vitro studies of antigen-specific T-cell responses in which the addition of anti-LAG-3 antibodies led to increased T-cell proliferation, higher expression of activation antigens such as CD25, and higher concentrations of cytokines such as interferon-gamma and interleukin-4 (Huard et al., Eur. J.
- CD4+CD25+ regulatory T-cells also have been shown to express LAG-3 upon activation and antibodies to LAG-3 inhibit suppression by induced Treg cells, both in vitro and in vivo, suggesting that LAG-3 contributes to the suppressor activity of Treg cells (Huang et al. Immunity, 21: 503-513 (2004)).
- LAG-3 has been shown to negatively regulate T-cell homeostasis by regulatory T-cells in both T-cell-dependent and independent mechanisms (Workman, C. J. and Vignali, D. A., J. Immunol., 174: 688-695 (2005)).
- LAG-3 activity is currently under investigation as a therapeutic approach to treat viral infections and melanoma based on preclinical studies.
- addition of soluble huLAG-3 fused to an Fc region enhanced the proliferation of antigen-specific T-cells to viral and tumor antigens, such as influenza matrix protein or melanoma antigen recognized by T-cells (MART-1), in PBMCs of healthy or cancer patients (Casati et al., J. Immunol, 180: 3782-3788 (2008)).
- LAG-3 e.g., an antibody
- the invention provides such LAG-3-binding agents.
- the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 Asp Ser Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
- the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Gln Val Gln Leu Gln Gln Trp Gly Ala Xaa1 Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Xaa2 Cys Xaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly Tyr Tyr Trp Xaa6 Trp e Arg Pro Xaa7 Lys Gly Leu Glu Trp e Gly Glu e Asn His Ser Gly Xaa8 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gln Xaa9 Ser Leu Lys Leu Xa10 Xaa11 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr Gly Asp Tyr Asp Tyr Trp Gly Gln Gly
- the invention further provides an isolated immunoglobulin heavy chain polypeptide comprising SEQ ID NO: 190 or 191.
- the invention provides an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Xaa1 Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Xaa2 Ser Gln Ser Leu Val His Ser Asp Xaa3 Xaa4 Thr Tyr Leu His Tip Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Xaa Xaa Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys e Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gln Ser Thr Xaa Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu He Lys Arg Thr (SEQ ID NO: 57), wherein (a) Xaa1
- the invention provides an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp e Ser Asn Tyr Leu Asn Trp Tyr Gin Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Val Tyr Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val (SEQ ID NO: 89), wherein (a
- the invention also provides isolated immunoglobulin light chain polypeptide comprising SEQ ID NO: 196 or 197.
- the invention provides isolated or purified nucleic acid sequences encoding the foregoing immunoglobulin polypeptides, vectors comprising such nucleic acid sequences, LAG-3-binding agents comprising the foregoing immunoglobulin polypeptides, nucleic acid sequences encoding such LAG-3-binding agents, vectors comprising such nucleic acid sequences, isolated cells comprising such vectors, compositions comprising such LAG-3-binding agents or such vectors with a pharmaceutically acceptable carrier, and methods of treating cancer or infectious diseases in mammals by administering effective amounts of such compositions to mammals.
- FIG. 1A is a graph of mean tumor volume over time in mice implanted with Colon26 colon adenocarcinoma cells and injected with the indicated antibodies. Each data plot in the figure refers to the indicated treatment group.
- FIG. 1B is a graph of tumor volume over time of individual animals in three treatment groups of mice implanted with Colon26 colon adenocarcinoma cells and injected with the indicated antibodies. Each data plot in the graphs refers to an individual animal in the treatment group.
- FIG. 2A depicts IL-2 secretion by CD4+ T-cells in a mixed lymphocyte reaction (MLR) assay at varying concentrations of Anti PD-1 or Anti-LAG-3 antibodies.
- MLR mixed lymphocyte reaction
- FIG. 2B depicts LAG-3 and PD-1 expression on CD4+ T-cells prior to (na ⁇ ve) or subsequent to (24, 48, and 72 hour) exposure to dendritic cells.
- the invention provides an isolated immunoglobulin heavy chain polypeptide and/or an isolated immunoglobulin light chain polypeptide, or a fragment (e.g., antigen-binding fragment) thereof.
- immunoglobulin or “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
- the polypeptide is “isolated” in that it is removed from its natural environment.
- an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR).
- the CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below).
- a whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
- Each of the heavy chains contains one N-terminal variable (V H ) region and three C-terminal constant (C H 1, C H 2, and C H 3) regions, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region.
- the light chains of antibodies can be assigned to one of two distinct types, either kappa ( ⁇ ) or lambda ( ⁇ ), based upon the amino acid sequences of their constant domains.
- each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds.
- the light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain.
- the remaining constant regions of the heavy chains are aligned with each other.
- variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
- the V H and V L regions have the same general structure, with each region comprising four framework (FW or FR) regions.
- framework region refers to the relatively conserved amino acid sequences within the variable region which are located between the hypervariable or complementary determining regions (CDRs).
- CDRs hypervariable or complementary determining regions
- the framework regions form the ⁇ sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5 th Ed ., Garland Publishing, New York, N.Y. (2001)).
- the framework regions are connected by three complementarity determining regions (CDRs).
- CDRs complementarity determining regions
- the three CDRs known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding.
- the CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions.
- the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions.
- the constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular toxicity via interactions with effector molecules and cells.
- the isolated immunoglobulin heavy chain polypeptide and the isolated immunoglobulin light chain polypeptide of the invention desirably bind to the protein encoded by the Lymphocyte Activation Gene-3 (LAG-3) (also referred to herein as “LAG-3 protein”).
- LAG-3 Lymphocyte Activation Gene-3
- LAG-3 is a 498 amino acid protein that negatively regulates T-cell function and homeostasis (Triebel et al., J. Exp. Med., 171(5): 1393-1405 (1990); and Triebel F., Trends Immunol., 24(12): 619-22 (2003)).
- LAG-3 is a member of the immunoglobulin supergene family and is structurally and genetically related to CD4.
- LAG-3 The intra-cytoplasmic region of LAG-3 has been shown to interact with a protein denoted LAP, which is thought to be a signal transduction molecule involved in the downregulation of the CD3/TCR activation pathway (Iouzalen et al., Eur. J. Immunol., 31: 2885-2891 (2001)). Furthermore, CD4+CD25+ regulatory T-cells (Treg) have been shown to express LAG-3 upon activation and antibodies to LAG-3 inhibit suppression by induced Treg cells, both in vitro and in vivo, suggesting that LAG-3 contributes to the suppressor activity of Treg cells (Huang et al., Immunity, 21: 503-513 (2004)).
- LAG-3 expression on CD4+ T-cells renders them more susceptible to suppression by Tregs, rather than making Tregs more suppressive (see Durham et al., PLoS ONE, 9(11): e109080 (2014)).
- LAG-3 also has been shown to have immunostimulatory effects (see, e.g., Prigent et al., Eur. J. Immunol., 29: 3867-3876 (1999)); El Mir and Triebel, J. Immunol., 164: 5583-5589 (2000)); and Casati et al., Cancer Res., 66: 4450-4460 (2006)).
- the inventive isolated immunoglobulin heavy chain polypeptide and the inventive isolated immunoglobulin light chain polypeptide can form an agent that binds to LAG-3 and another antigen, resulting in a “dual reactive” binding agent (e.g., a dual reactive antibody).
- a dual reactive binding agent e.g., a dual reactive antibody
- the agent can bind to LAG-3 and to another negative regulator of the immune system such as, for example, programmed death 1 (PD-1) and/or T-cell immunoglobulin domain and mucin domain 3 protein (TIM-3).
- PD-1 programmed death 1
- TIM-3 T-cell immunoglobulin domain and mucin domain 3 protein
- Antibodies which bind to LAG-3, and components thereof, are known in the art (see, e.g., U.S. Patent Application Publication Nos. 2010/0233183, 2011/0150892, and 2014/0093511).
- Anti-LAG-3 antibodies also are commercially available from sources such as, for example, Abcam (Cambridge, Mass.), and R&D Systems, Inc. (Minneapolis, Minn.).
- the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 Asp Ser Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
- the immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of the amino acid sequence Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr e His Trp Val Xaa3 Gin Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp e Asp Xaa5 Glu Asn Xaa6 Asp Ser Glu Tyr Xaa7 Ser Lys Phe Xaa8 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa9 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Thr Val Ser Ser (SEQ ID NO:
- the isolated immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 10,
- the invention also provides an immunoglobulin heavy chain polypeptide that comprises, consists of, or consists essentially of the amino acid sequence Gin Val Gin Leu Gin Gin Trp Gly Ala Xaa1 Leu Leu Lys Pro Ser Glu Thr Lu Ser Leu Xaa2 Cys Xaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly Tyr Tyr Trp Xaa6 Trp Ile Arg Gin Pro Pro Pro Xaa7 Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Xaa8 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr e Ser Val Asp Thr Ser Lys Asn Gin Xaa9 Ser Leu Lys Leu Xaa10 Xaa11 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Tyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr Gly Asp Tyr Asp Tyr Trp Gly
- the isolated immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.
- an isolated immunoglobulin heavy chain polypeptide which comprises SEQ ID NO: 190 or 191.
- examples of such a polypeptide include those comprising any one of SEQ ID NOs: 192-195.
- inventive immunoglobulin heavy chain polypeptide consists essentially of an amino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 56, SEQ ID NOS: 182-186, or SEQ ID NOS: 190-195
- additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
- the inventive immunoglobulin heavy chain polypeptide consists of an amino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 56
- the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
- the invention provides an isolated immunoglobulin heavy chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 1-56.
- Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence.
- the percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer).
- a number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences).
- BLAST programs e.g., BLAST 2.1, BL2SEQ, and later versions thereof
- FASTA programs e.g., FASTA3x, FASTM, and SSEARCH
- Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci.
- the invention provides an immunoglobulin light chain polypeptide that comprises, consists of, or consists essentially of, an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Xaa1 Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gin Pro Ala Ser Ile Ser Cys Arg Xaa2 Ser Gln Ser Leu Val His Ser Asp Xaa3 Xaa4 Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gin Ser Pro Gin Leu Leu Ile Tyr Xaa Xaa Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gin Ser Thr Xaa Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
- the isolated immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61.
- SEQ ID NO: 74. SEQ ID NO: 75, SEQ ID NO: 76.
- the invention provides an isolated immunoglobulin light chain polypeptide which comprises, consists essentially of, or consists of the amino acid sequence Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Val Tyr Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
- the inventive immunoglobulin light chain polypeptide can include or lack the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 at positions 49-53 of SEQ ID NO: 89 when Xaa6 is threonine (Thr) or isoleucine (Ile).
- Xaa6 is threonine (Thr) or isoleucine (Ile).
- inventive immunoglobulin light chain polypeptide comprises the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5
- each of Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 can be any suitable amino acid residue.
- Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 can be any suitable amino acid residue.
- Xaa1 is tyrosine (Tyr), Xaa2 is aspartic acid (Asp).
- Xaa3 is alanine (Ala), Xaa4 is serine (Ser), and Xaa5 is asparagine (Asn).
- a preferred amino acid sequence of an immunoglobulin light chain polypeptide which includes the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 comprises SEQ ID NO: 90.
- the immunoglobulin light chain polypeptide When the immunoglobulin light chain polypeptide lacks the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5, the immunoglobulin light chain polypeptide preferably comprises the amino acid sequence SEQ ID NO: 91 or SEQ ID NO: 92.
- an isolated immunoglobulin light chain polypeptide which comprises SEQ ID NO: 196 or 197.
- examples of such a polypeptide include those comprising any one of SEQ ID NOs: 198-200.
- inventive immunoglobulin light chain polypeptide consists essentially of an amino acid sequence of any one of SEQ ID NO: 57-SEQ ID NO: 92, SEQ ID NOs: 187-189, or SEQ ID NOs: 196-200
- additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation).
- the inventive immunoglobulin light chain polypeptide consists of an amino acid sequence of any one of SEQ ID NO: 57-SEQ ID NO: 92
- the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin light chain polypeptide).
- the invention provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 57-SEQ ID NO: 92.
- Nucleic acid or amino acid sequence “identity” can be determined using the methods described herein.
- One or more amino acids of the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides can be replaced or substituted with a different amino acid.
- An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
- Amino acids are broadly grouped as “aromatic” or “aliphatic.”
- An aromatic amino acid includes an aromatic ring.
- aromatic amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp).
- Non-aromatic amino acids are broadly grouped as “aliphatic.”
- “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).
- Aliphatic amino acids may be sub-divided into four sub-groups.
- the “large aliphatic non-polar sub-group” consists of valine, leucine, and isoleucine.
- the “aliphatic slightly-polar sub-group” consists of methionine, serine, threonine, and cysteine.
- the “aliphatic polar/charged sub-group” consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine.
- the “small-residue sub-group” consists of glycine and alanine.
- the group of charged/polar amino acids may be sub-divided into three sub-groups: the “positively-charged sub-group” consisting of lysine and arginine, the “negatively-charged sub-group” consisting of glutamic acid and aspartic acid, and the “polar sub-group” consisting of asparagine and glutamine.
- Aromatic amino acids may be sub-divided into two sub-groups: the “nitrogen ring sub-group” consisting of histidine and tryptophan and the “phenyl sub-group” consisting of phenylalanine and tyrosine.
- the amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative.
- the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
- a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure , Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
- conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free —OH can be maintained, and glutamine for asparagine such that a free —NH 2 can be maintained.
- “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
- one or more amino acids can be inserted into the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides. Any number of any suitable amino acids can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide. In this respect, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide.
- at least one amino acid e.g., 2 or more, 5 or more, or 10 or more amino acids
- 20 amino acids e.g., 18 or less, 15 or less, or 12 or less amino acids
- 1-10 amino acids are inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide.
- the amino acid(s) can be inserted into any one of the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides in any suitable location.
- the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide.
- inventive isolated immunoglobulin heavy chain polypeptide and light chain polypeptides are not limited to polypeptides comprising the specific amino acid sequences described herein.
- the immunoglobulin heavy chain polypeptide or light chain polypeptide can be any heavy chain polypeptide or light chain polypeptide that competes with the inventive immunoglobulin heavy chain polypeptide or light chain polypeptide for binding to LAG-3.
- the immunoglobulin heavy chain polypeptide or light chain polypeptide can be any heavy chain polypeptide or light chain polypeptide that binds to the same epitope of LAG-3 recognized by the heavy and light chain polypeptides described herein.
- Antibody competition can be assayed using routine peptide competition assays which utilize ELISA, Western blot, or immunohistochemistry methods (see, e.g., U.S. Pat. Nos. 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4: 12 (2006)).
- the invention provides an isolated LAG-3-binding agent comprising, consisting essentially of, or consisting of one or more of the inventive isolated amino acid sequences described herein.
- LAG-3-binding agent is meant a molecule, preferably a proteinaceous molecule, which binds specifically to the LAG-3 protein.
- the LAG-3-binding agent is an antibody or a fragment (e.g., immunogenic fragment) thereof.
- the LAG-3-binding agent of the invention comprises, consists essentially of, or consists of the inventive isolated immunoglobulin heavy chain polypeptide and/or the inventive isolated immunoglobulin light chain polypeptide.
- the LAG-3-binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide or the inventive immunoglobulin light chain polypeptide. In another embodiment, the LAG-3-binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide and the inventive immunoglobulin light chain polypeptide.
- any amino acid residue of the inventive immunoglobulin heavy chain polypeptide and/or the inventive immunoglobulin light chain polypeptide can be replaced, in any combination, with a different amino acid residue, or can be deleted or inserted, so long as the biological activity of the LAG-3-binding agent is enhanced or improved as a result of the amino acid replacements, insertions, and/or deletions.
- the “biological activity” of an LAG-3-binding agent refers to, for example, binding affinity for a particular LAG-3 epitope, neutralization or inhibition of LAG-3 binding to its receptor(s), neutralization or inhibition of LAG-3 activity in vivo (e.g., IC 50 ), pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the LAG-3 protein, or with other proteins or tissues).
- Other biological properties or characteristics of an antigen-binding agent recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, and formulation.
- the aforementioned properties or characteristics can be observed, measured, and/or assessed using standard techniques including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORETM), or KINEXATM, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and/or secretion assays, and signal transduction and immunohistochemistry assays.
- standard techniques including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORETM), or KINEXATM, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and/or secretion assays, and signal transduction and immunohistochemistry assays.
- the terms “inhibit” or “neutralize,” as used herein with respect to the activity of a LAG-3-binding agent, refer to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the biological activity of LAG-3, or a disease or condition associated with LAG-3.
- the isolated LAG-3-binding agent of the invention preferably inhibits or neutralizes the activity of LAG-3 by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the foregoing values.
- the isolated LAG-3-binding agent of the invention can be a whole antibody, as described herein, or an antibody fragment.
- fragment of an antibody “antibody fragment,” and “functional fragment of an antibody” are used interchangeably herein to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)).
- the isolated LAG-3-binding agent can contain any LAG-3-binding antibody fragment.
- the antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof.
- antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the V L , V H , C L , and CH 1 domains, (ii) a F(ab′) 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) a Fab′ fragment, which results from breaking the disulfide bridge of an F(ab′) 2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
- a Fab fragment which is a monovalent fragment consisting of the V L , V H , C L , and CH 1 domains
- the isolated LAG-3-binding agent comprises a fragment of the immunoglobulin heavy chain or light chain polypeptide
- the fragment can be of any size so long as the fragment binds to, and preferably inhibits the activity of, LAG-3.
- a fragment of the immunoglobulin heavy chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids.
- a fragment of the immunoglobulin light chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids.
- the LAG-3-binding agent is an antibody or antibody fragment
- the antibody or antibody fragment desirably comprises a heavy chain constant region (Fe) of any suitable class.
- the antibody or antibody fragment comprises a heavy chain constant region that is based upon wild-type IgG1, IgG2, or IgG4 antibodies, or variants thereof.
- the LAG-3 binding agent comprises an Fc region engineered to reduce or eliminate effector functions of the antibody.
- Engineered Fc regions with reduced or abrogated effector function are known in the art and commercially available, as are techniques for engineering Fc regions to reduce or eliminate effector function, any of which can be used in conjunction with the invention.
- the LAG-3-binding agent also can be a single chain antibody fragment.
- single chain antibody fragments include, but are not limited to, (i) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., V L and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat.
- scFv single chain Fv
- a diabody which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a V H connected to a V L by a peptide linker that is too short to allow pairing between the V H and V L on the same polypeptide chain, thereby driving the pairing between the complementary domains on different V H -V L polypeptide chains to generate a dimeric molecule having two functional antigen binding sites.
- Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009/0093024 A1.
- the isolated LAG-3-binding agent also can be an intrabody or fragment thereof.
- An intrabody is an antibody which is expressed and which functions intracellularly. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated V H and V L domains and scFvs.
- An intrabody can include sub-cellular trafficking signals attached to the N or C terminus of the intrabody to allow expression at high concentrations in the sub-cellular compartments where a target protein is located.
- an intrabody Upon interaction with a target gene, an intrabody modulates target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment.
- Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in protein-protein, protein-DNA, or protein-RNA interactions.
- the isolated LAG-3-binding agent also can be an antibody conjugate.
- the isolated LAG-3-binding agent can be a conjugate of (1) an antibody, an alternative scaffold, or fragments thereof, and (2) a protein or non-protein moiety comprising the LAG-3-binding agent.
- the LAG-3-binding agent can be all or part of an antibody conjugated to a peptide, a fluorescent molecule, or a chemotherapeutic agent.
- the isolated LAG-3-binding agent can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody.
- chimeric is meant an antibody or fragment thereof comprising both human and non-human regions.
- the isolated LAG-3-binding agent is a humanized antibody.
- a “humanized” antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody.
- Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat).
- a humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody.
- CDRH3 of the inventive LAG-3-binding agent is obtained or derived from a mouse monoclonal antibody, while the remaining variable regions and constant region of the inventive LAG-3-binding agent are obtained or derived from a human monoclonal antibody.
- a human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents).
- in vitro sources e.g., a hybridoma or a cell line producing an antibody recombinantly
- in vivo sources e.g., rodents.
- a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes.
- transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSETM, the Kirin TC MOUSETM, and the Kyowa Kirin KM-MOUSETM (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)).
- a humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic , John Wiley & Sons. Inc., Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115-120 (2008)).
- a humanized antibody can be produced using the methods described in, e.g., U.S. Patent Application Publication 2011/0287485 A1.
- a CDR e.g., CDR1, CDR2, or CDR3
- a variable region of the immunoglobulin heavy chain polypeptide and/or the immunoglobulin light chain polypeptide described herein can be transplanted (i.e., grafted) into another molecule, such as an antibody or non-antibody polypeptide, using either protein chemistry or recombinant DNA technology.
- the invention provides an isolated LAG-3-binding agent comprising at least one CDR of an immunoglobulin heavy chain and/or light chain polypeptide as described herein.
- the isolated LAG-3-binding agent can comprise one, two, or three CDRs of an immunoglobulin heavy chain and/or light chain variable region as described herein.
- the LAG-3-binding agent binds an epitope of LAG-3 which blocks the binding of LAG-3 to MHC Class II molecules and inhibits LAG-3-mediated signaling.
- the LAG-3 binding agent can bind to one or more of the four Ig-like extracellular domains (D1-D4) of the LAG-3 protein (see, e.g, Triebel et al., J. Exp. Med., 171(5): 1393-1405 (1990); and Bruniquel et al., Immunogenetics, 47: 96-98 (1997)).
- the LAG-3 binding agent binds to domain 1 (D1) and/or domain (D2) of the LAG-3 protein.
- the invention also provides an isolated or purified epitope of LAG-3 which blocks the binding of LAG-3 to MHC Class II molecules in an indirect or allosteric manner.
- the invention also provides one or more isolated or purified nucleic acid sequences that encode the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and the inventive LAG-3-binding agent.
- nucleic acid sequence is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.
- nucleic acid and polynucleotide refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA.
- RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.
- Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).
- the invention further provides a vector comprising one or more nucleic acid sequences encoding the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive LAG-3-binding agent.
- the vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage.
- Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3 rd edition , Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology , Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).
- the vector preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., the osteonectin signal peptide), internal ribosome entry sites (IRES), and the like, that provide for the expression of the coding sequence in a host cell.
- expression control sequences such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., the osteonectin signal peptide), internal ribosome entry sites (IRES), and the like.
- Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology , Vol. 185, Academic Press, San Diego, Calif. (1990).
- promoters including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art.
- Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources.
- Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction).
- Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter.
- Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad.
- Enhancers refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences.
- the vector also can comprise a “selectable marker gene.”
- selectable marker gene refers to a nucleic acid sequence that allow cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and described in, e.g., International Patent Application Publications WO 1992/008796 and WO 1994/028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Aced. Sci. USA, 78: 1527-1531(1981); Mulligan & Berg, Proc. Natl.
- the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11: 1735-1742 (2004)).
- Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP).
- the vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP.
- Suitable vectors include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome. Such integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a site specific manner include, for example, components of the fip-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNATM5/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, Calif.).
- vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Life Technologies (Carlsbad, Calif.), UCOE from Millipore (Billerica, Mass.), and pCI or pFN10A (ACT) FLEXITM from Promega (Madison, Wis.).
- Viral vectors also can be used.
- Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentiviral-based pLP1 from Invitrogen (Carlsbad, Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.).
- Nucleic acid sequences encoding the inventive amino acid sequences can be provided to a cell on the same vector (i.e., in cis).
- a unidirectional promoter can be used to control expression of each nucleic acid sequence.
- a combination of bidirectional and unidirectional promoters can be used to control expression of multiple nucleic acid sequences.
- Nucleic acid sequences encoding the inventive amino acid sequences alternatively can be provided to the population of cells on separate vectors (i.e., in trans). Each of the nucleic acid sequences in each of the separate vectors can comprise the same or different expression control sequences. The separate vectors can be provided to cells simultaneously or sequentially.
- the vector(s) comprising the nucleic acid(s) encoding the inventive amino acid sequences can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby, including any suitable prokaryotic or eukaryotic cell.
- the invention provides an isolated cell comprising the inventive vector.
- Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently.
- suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis ), Escherichia (such as E. coli ), Pseudomonas, Streptomyces, Salmonella , and Erwinia .
- Particularly useful prokaryotic cells include the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).
- the vector is introduced into a eukaryotic cell.
- Suitable eukaryotic cells include, for example, yeast cells, insect cells, and mammalian cells.
- suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino - sporidium, Saccharomyces , and Schizosaccharomyces .
- Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.
- Suitable insect cells are described in, for example, Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4: 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993).
- Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).
- mammalian cells are utilized in the invention.
- suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.).
- suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO)(ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 42164220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92).
- CHO Chinese hamster ovary cells
- CHO DHFR-cells Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 42164220 (1980)
- human embryonic kidney (HEK) 293 or 293T cells ATCC No. CRL1573)
- 3T3 cells
- mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
- Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Methods for selecting suitable mammalian host cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.
- the mammalian cell is a human cell.
- the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin.
- human lymphoid cells lines include, without limitation, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85:1581-1585 (1988)), Raji cells (CCL-86), PER.C6 cells (Crucell Holland B.V., Leiden, The Netherlands), and derivatives thereof.
- a nucleic acid sequence encoding the inventive amino acid sequence may be introduced into a cell by “transfection,” “transformation,” or “transduction.”
- “Transfection,” “transformation.” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.
- Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol.
- Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
- the invention provides a composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence.
- the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector.
- a pharmaceutically acceptable composition which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector.
- Any suitable carrier can be used within the context of the invention, and such carriers are well known in the art.
- the choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition.
- the composition optionally can be sterile.
- the composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use.
- the compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21 st Edition , Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).
- the invention further provides a method of treating a disorder in a mammal that is responsive to LAG-3 inhibition or neutralization.
- the method comprises administering the aforementioned composition to a mammal having a disorder that is responsive to LAG-3 inhibition or neutralization, whereupon the disorder is treated in the mammal.
- a disorder that is “responsive to LAG-3 inhibition” or “responsive to LAG-3 neutralization” refers to any disease or disorder in which a decrease in LAG-3 levels or activity has a therapeutic benefit in mammals, preferably humans, or the improper expression (e.g., overexpression) or increased activity of LAG-3 causes or contributes to the pathological effects of the disease or disorder.
- Disorders that are responsive to LAG-3 inhibition include, for example, cancer and infectious diseases.
- the inventive method can be used to treat any type of cancer known in the art, such as, for example, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma (see, e.g., Bhatia et al., Curr. Oncol. Rep., 13(6): 488-497 (2011)).
- the inventive method can be used to treat any type of infectious disease (i.e., a disease or disorder caused by a bacterium, a virus, a fungus, or a parasite).
- infectious diseases examples include, but are not limited to, diseases caused by a human immunodeficiency virus (HIV), a respiratory syncytial virus (RSV), an influenza virus, a dengue virus, a hepatitis B virus (HBV, or a hepatitis C virus (HCV)).
- HCV human immunodeficiency virus
- RSV respiratory syncytial virus
- influenza virus a dengue virus
- HBV hepatitis B virus
- HCV hepatitis C virus
- Administration of a composition comprising the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence induces an immune response against a cancer or infectious disease in a mammal.
- An “immune response” can entail, for example, antibody production and/or the activation of immune effector cells (
- the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect.
- the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease.
- the inventive method comprises administering a “therapeutically effective amount” of the LAG-3-binding agent.
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
- the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the LAG-3-binding agent to elicit a desired response in the individual.
- a therapeutically effective amount of a LAG-3-binding agent of the invention is an amount which decreases LAG-3 bioactivity in a human.
- the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof.
- the inventive method comprises administering a “prophylactically effective amount” of the LAG-3-binding agent.
- a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).
- a typical dose can be, for example, in the range of 1 ⁇ g/kg to 20 mg/kg of animal or human body weight; however, doses below or above this exemplary range are within the scope of the invention.
- the daily parenteral dose can be about 0.00001 ⁇ g/kg to about 20 mg/kg of total body weight (e.g., about 0.001 ⁇ g/kg, about 0.1 ⁇ g/kg, about 1 ⁇ g/kg, about 5 ⁇ g/kg, about 10 ⁇ g/kg, about 100 ⁇ g/kg, about 500 ⁇ g/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two of the foregoing values), preferably from about 0.1 ⁇ g/kg to about 10 mg/kg of total body weight (e.g., about 0.5 ⁇ g/kg, about 1 ⁇ g/kg, about 50 ⁇ g/kg, about 150 ⁇ g/kg, about 300 ⁇ g/kg, about 750 ⁇ g/kg,
- Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the invention.
- the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
- composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
- the composition preferably is suitable for parenteral administration.
- parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
- the biological activity of the inventive LAG-3-binding agent can be measured by any suitable method known in the art.
- the biological activity can be assessed by determining the stability of a particular LAG-3-binding agent.
- the LAG-3-binding agent e.g., an antibody
- the LAG-3-binding agent has an in vivo half life between about 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values).
- the LAG-3-binding agent has an in vivo half life between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values).
- the LAG-3-binding agent has an in vivo half life between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).
- the biological activity of a particular LAG-3-binding agent also can be assessed by determining its binding affinity to LAG-3 or an epitope thereof.
- affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ).
- Affinity of a binding agent to a ligand, such as affinity of an antibody for an epitope can be, for example, from about 1 picomolar (pM) to about 100 micromolar ( ⁇ M) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1 micromolar ( ⁇ M), or from about 1 ⁇ M to about 100 ⁇ M).
- the LAG-3-binding agent can bind to an LAG-3protein with a K D less than or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values).
- 1 nanomolar e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values).
- the LAG-3-binding agent can bind to LAG-3 with a K D less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values).
- Immunoglobulin affinity for an antigen or epitope of interest can be measured using any art-recognized assay.
- Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXATM), antigen panning, and/or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 2001).
- FACS fluorescence activated cell sorting
- separable beads e.g., magnetic beads
- SPR surface plasmon resonance
- KINEXATM solution phase competition
- the LAG-3-binding agent of the invention may be administered alone or in combination with other drugs (e.g., as an adjuvant).
- the LAG-3-binding agent can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein.
- the LAG-3-binding agent can be used in combination with at least one other anticancer agent including, for example, any chemotherapeutic agent known in the art, ionization radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer), and/or surgery.
- the LAG-3-binding agent can be administered in combination with at least one anti-bacterial agent or at least one anti-viral agent.
- the anti-bacterial agent can be any suitable antibiotic known in the art.
- the anti-viral agent can be any vaccine of any suitable type that specifically targets a particular virus (e.g., live-attenuated vaccines, subunit vaccines, recombinant vector vaccines, and small molecule anti-viral therapies (e.g., viral replication inhibitors and nucleoside analogs).
- the inventive LAG-3 binding agent can be administered in combination with other agents that inhibit immune checkpoint pathways.
- the inventive LAG-3 binding agent can be administered in combination with agents that inhibit or antagonize the programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 protein (TIM-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways.
- PD-1 programmed death 1
- TIM-3 T-cell immunoglobulin domain and mucin domain 3 protein
- CTLA-4 cytotoxic T-lymphocyte-associated protein 4
- the inventive LAG-3 binding agent is administered in combination with an antibody that binds to TIM-3 and/or an antibody that binds to PD-1.
- the inventive method of treating a cancer or an infectious disease in a mammal can further comprise administering to the mammal a composition comprising (i) an antibody that binds to a TIM-3 protein and (ii) a pharmaceutically acceptable carrier or a composition comprising (i) an antibody that binds to a PD-1 protein and (ii) a pharmaceutically acceptable carrier.
- the LAG-3-binding agent described herein can be used in diagnostic or research applications.
- the LAG-3-binding agent can be used in a method to diagnose a disorder or disease in which the improper expression (e.g., overexpression) or increased activity of LAG-3 causes or contributes to the pathological effects of the disease or disorder.
- the LAG-3-binding agent can be used in an assay to monitor LAG-3 protein levels in a subject being tested for a disease or disorder that is responsive to LAG-3 inhibition.
- Research applications include, for example, methods that utilize the LAG-3-binding agent and a label to detect an LAG-3 protein in a sample. e.g., in a human body fluid or in a cell or tissue extract.
- the LAG-3-binding agent can be used with or without modification, such as covalent or non-covalent labeling with a detectable moiety.
- the detectable moiety can be a radioisotope (e.g., 3 H, 14 C, 32 P, 35 S, or 125 I), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), or prosthetic groups.
- a radioisotope e.g., 3 H, 14 C, 32 P, 35 S, or 125 I
- a fluorescent or chemiluminescent compound e.g., fluorescein isothiocyanate, rhodamine, or luciferin
- an enzyme e.g., alkaline phosphatase, beta-galacto
- any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed in the context of the invention (see, e.g., Hunter et al., Nature, 194: 495-496 (1962); David et al., Biochemistry, 13: 1014-1021(1974); Pain et al., J. Immunol. Meth., 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982)).
- LAG-3 protein levels can be measured using the inventive LAG-3-binding agent by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA), and FACS.
- RIA radioimmunoassay
- Normal or standard expression values of LAG-3 can be established using any suitable technique, e.g., by combining a sample comprising, or suspected of comprising, LAG-3 with a LAG-3-specific antibody under conditions suitable to form an antigen-antibody complex.
- the antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody.
- Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc. (1987)). The amount of LAG-3 polypeptide expressed in a sample is then compared with a standard value.
- the LAG-3-binding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a diagnostic assay.
- the kit desirably includes substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides a detectable chromophore or fluorophore).
- substrates and cofactors required by the enzyme e.g., a substrate precursor which provides a detectable chromophore or fluorophore.
- other additives may be included in the kit, such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer), and the like.
- the relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
- the reagents may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate
- This example demonstrates a method of generating monoclonal antibodies directed against human LAG-3.
- mice The gene encoding the extracellular domain (ECD) of human LAG-3 was fused to either mouse IgG2a (human LAG-3 mIgG2a Fc) or a disabled form of wasabi fluorescent protein (dWFP human LAG-3) to produce antigen for use in mouse immunization and hybridoma screening.
- mouse IgG2a human LAG-3 mIgG2a Fc
- dWFP human LAG-3 a disabled form of wasabi fluorescent protein
- mice were purchased from Harlan Laboratories, Inc. (Indianapolis, Ind.) and divided into two groups.
- one group of animals was immunized with four to six doses of purified human LAG-3 mIgG2a Fc at 50 ⁇ g/mouse at intervals of three to four weeks using complete Freund's adjuvant (CFA) or incomplete Freunds adjuvant (IFA).
- CFA complete Freund's adjuvant
- IFA incomplete Freunds adjuvant
- the second group of animals was injected with four to six doses at intervals of three to four weeks alternating between human LAG-3 mIgG2a F or dWFP human LAG-3 ECD.
- CFA or IFA was also used as adjuvant in the second group. Animals were bled for measurement of the serum titer to human LAG-3 as assessed by binding to cell surface human LAG-3.
- CHO-S cells were transfected with a full length human LAG-3 extracellular domain fused to the H-2Kk transmembrane domain (CHO-S huLAG-3 ECD cells). Sera were diluted from 1:1,000-1:1,000,000 and incubated with the CHO-S huLAG-3 ECD cells for 30 minutes at 4° C. Cells were centrifuged, washed once with PBS/1% BSA, and incubated with PE-conjugated (Southern Biotech, Birmingham, Ala.) or ALEXAFLUORTM 647- (Jackson Immunoresearch, West Grove, Pa.) labeled goat anti-mouse IgG (H+L) for 30 minutes at 4° C.
- PE-conjugated Southernn Biotech, Birmingham, Ala.
- ALEXAFLUORTM 647- Jackson Immunoresearch, West Grove, Pa.
- Hybridoma supernatants were screened for binding to CHO-S huLAG-3 ECD cells and compared to binding to untransfected CHO-S cells as described above. Based upon binding CHO-S huLAG-3 ECD cells, hybridomas were transferred to 48-well plates and expanded.
- the results of this example confirm the production of anti-LAG-3 monoclonal antibodies using hybridoma cell technology.
- This example describes the design and generation of CDR-grafted and chimeric anti-LAG-3 monoclonal antibodies.
- Antibodies from the hybridomas described in Example 1 were isotyped, subjected to RT-PCR for cloning the antibody heavy chain variable region (VH) and light chain variable region (VL), and sequenced. Specifically, RNA was isolated from cell pellets of hybridoma clones (1 ⁇ 10 6 cells/pellet) using the RNEASYTM kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using oligo-dT-primed SUPERSCRIPTTM III First-Strand Synthesis System (Life Technologies, Carlsbad, Calif.).
- PCR amplification of the VL utilized a pool of degenerate mouse V L forward primers (see Kontermann and Dubel, eds., Antibody Engineering , Springer-Verlag, Berlin (2001)) and a mouse a constant region reverse primer.
- PCR amplification of the VH utilized a pool of degenerate mouse VH forward primers (Kontermann and Dubel, supra) and a mouse ⁇ 1 or ⁇ 2a constant region reverse primer (based on isotyping of purified antibody from each clone) with the protocol recommended in the SUPERSCRIPTTM III First-Stand Synthesis System (Life Technologies, Carlsbad, Calif.). PCR products were purified and cloned into pcDNA3.3-TOPO (Life Technologies, Carlsbad, Calif.).
- CDR-grafted antibody sequences were designed by cloning CDR residues from each of the above-described mouse antibodies into the closest human germline homolog. CDR-grafted antibody variable regions were synthesized and expressed with human IgG/ ⁇ constant regions for analysis. In addition, mouse:human chimeric antibodies were constructed using the variable regions of the above-described mouse antibodies linked to human IgG/ ⁇ constant regions. Chimeric and CDR-grafted antibodies were characterized for binding to CHO-S huLAG-3 ECD cells and for activity in the human LAG-3 ECD/Daudi blocking assay as described above.
- the functional antagonist activity of chimeric and CDR-grafted antibodies also was tested in a human CD4 ⁇ T-cell:dendritic cell mixed lymphocyte reaction (MLR) assay in which activation of CD4 ⁇ T-cells in the presence of anti-LAG-3 antibodies is assessed by measuring IL-2 secretion.
- MLR human CD4 ⁇ T-cell:dendritic cell mixed lymphocyte reaction
- antagonism of LAG-3 was expected to result in increased T-cell activation as measured by increased IL-2 production.
- the 5.B11, 5.D7, and 1.E10 CDR-grafted antibodies demonstrated antagonistic activity in the MLR assay as measured by an increase in IL-2 activity.
- results of this example demonstrate a method of generating chimeric and CDR-grafted monoclonal antibodies that specifically bind to and inhibit LAG-3.
- This example demonstrates affinity maturation of humanized monoclonal antibodies directed against human LAG-3.
- iSHM silico somatic hypermutation
- This method incorporates mutations as predicted by computational analysis comparing in vivo matured antibody sequences, as downloaded from NCBI, and comparing them to germline human IGHV, IGKV, and IGLV sequences and their allelic forms (as described in Bowers et al., J. Biol. Chem., 288(11):7688-7696 (2013)).
- the LAG-3 binding properties of resultant antibodies were assayed using surface plasmon resonance (SPR) as well as ability to bind to CHO-S huLAG-3 ECD cells as described previously.
- Solution-based affinity analyses were also performed on using a KINEXATM 3000 assay (Sapidyne Instruments, Boise, Id.), and results were analyzed using KINEXATM Pro Software 3.2.6. Experimental parameters were selected to reach a maximum signal with antibody alone between 0.8 and 1.2 V, while limiting nonspecific binding signal with buffer alone to less than 10% of the maximum signal.
- Azlactone beads (50 mg) were coated with antigen by diluting in a solution of human or cynoWFP-LAG-3 (50 ⁇ g/mL in 1 mL) in 50 mM Na 2 CO 3 .
- the solution was rotated at room temperature for 2 hours, and beads were pelleted in a picofuge and washed twice with blocking solution (10 mg/mL BSA, 1 M Tris-HCl, pH 8.0). Beads were resuspended in blocking solution (1 mL), rotated at room temperature for 1 hour, and diluted in 25 volumes PBS/0.02% NaN 3 .
- the secondary antibody was ALEXFLUORTM 647 dye-anti-human IgG (500 ng/mL).
- Sample antibody concentrations were held constant (50 pM or 75 pM), while human or cynomolgus WFP-LAG-3 antigen was titrated using a three-fold dilutions series from 1 ⁇ M to 17 pM. All samples were diluted in PBS, 0.2% NaN 3 , 1 mg/mL BSA and allowed to equilibrate at room temperature for 30 hours. Additionally, samples containing only antibody and only buffer were tested in order to determine maximum signal and nonspecific binding signal, respectively.
- Thermal stability of the selected antibodies was assessed using a Thermofluor assay as described in McConnell et al., Protein Eng. Des. Sel., 26: 151 (2013). This assay assesses stability through the ability of a hydrophobic fluorescent dye to bind to hydrophobic patches on the protein surface which are exposed as the protein unfolds. The temperature at which 50% of the protein unfolds (Tm) is determined to measure thermal stability. This assay demonstrated that 5.D7 monoclonal antibody variants had acceptable melting temperatures (Tms) (i.e., greater than 70° C.) that were suitable for drug development.
- Tms melting temperatures
- the results of this example confirm a method of affinity maturing humanized monoclonal antibodies directed against LAG-3.
- This example demonstrates a method of identifying antibodies directed against human LAG-3 from an evolvable library.
- IgG evolvable library based on germline sequence V-gene segments joined to human donor-derived recombined (D)J regions, was constructed as described in Bowers et al. Proc. Natl. Acad Sci. USA, 108(51): 20455-20460 (2011).
- IgG heavy chain (HC) and light chain (LC) were cloned into separate episomal vectors (Horlick et al., Gene, 243(1-2): 187-194 (2000)), with each vector encoding a distinct antibiotic selectable marker.
- the HC vector was formatted such that antibody was presented both on the cell surface as well as secreted into the tissue culture medium (Horlick et al., J. Biol.
- HCs and LCs were co-transfected into HEK293 cells and expanded to approximately 10 cells.
- the cell library was then subjected to two rounds each of negative selection against streptavidin (SA)-coupled magnetic beads alone (catalog #11047, Life Technologies, Carlsbad, Calif.) and irrelevant biotinylated antigen coated with SA-coupled magnetic beads.
- SA streptavidin
- One round of positive selection was then performed using either magnetic beads coated directly with human LAG-3 mIgG2a Fc or with SA-coupled magnetic beads coated with biotinylated LAG-3 ECD mIgG1 Fc.
- the positively selected cells were diluted and plated in 96-well format at an approximate density of 1-10 cells/well. Resulting colonies were expanded into daughter plates and a portion of each population was tested for binding to LAG-3 ECD mIgG1 Fc DyL650 by FACSARRAYTM analysis. Antibodies secreted into the supernatant also were tested by BIACORETM for ability to bind to LAG-3 ECD mIgG1 Fc.
- Antibodies also were characterized for their ability to bind to cynomolgus monkey LAG-3 protein (cyno LAG-3). As these germline antibodies identified from the library were too weak to bind to antigen expressed on the cell surface, soluble antigen similar to the human antigen was labeled with DyL650 (cyno LAG-3 mIgG2a Fc DyL650) and then incubated with HEK293 cells displaying antibody strategies on the cell surface. Eight antibody strategies identified from the evolvable library were tested and demonstrated an ability to bind to cyno LAG-3 ECD mIgG1 Fc.
- results of this example confirm that monoclonal antibodies directed against human and non-human LAG-3 can be identified using an evolvable library.
- This example demonstrates affinity maturation of antibodies directed against human LAG-3 identified using an evolvable library.
- Stable cell lines co-expressing the HC and LC of each antibody identified from the evolvable library described in Example 4 were transfected with activation induced cytidine deaminase (AID) to initiate in vitro SHM.
- AID was also transfected directly into the original mixed population of cells expanded from the library screen. In all cases, cell populations were stained for both IgG expression and binding to antigen, collected by flow cytometry as a bulk population, and then expanded for sequence analysis by next generation sequencing (NGS). This process was repeated iteratively to accumulate SHM-derived mutations in the variable regions of both the heavy and light chains, and their derivatives, for each strategy.
- NGS next generation sequencing
- Thermal stability of the selected antibodies was assessed using a Thermofluor assay as described above. This assay demonstrated that select monoclonal antibodies from the A17 strategy had acceptable T m s that were suitable for drug development. Antibodies also were tested for binding to HEK 293f cells using a flow cytometry-based assay. The results indicated that non-specific binding was low for select A17 candidates.
- results of this example confirm a method of affinity maturing monoclonal antibodies directed against LAG-3 identified using an evolvable library.
- an inventive anti-LAG-3 monoclonal antibody can inhibit LAG-3 signaling and enhance T-cell activation in vitro alone and in combination with an anti-PD-1 antibody or an anti-TIM-3 antibody.
- the anti-PD-1 antibody APE02058 was titrated in a dose-response in the human CD4+ T-cell MLR assay described above. Based on the results from titrating the anti-PD-1 antibody in multiple MLR assays, 133 pM (approximate EC50) and 13 pM (approximate EC10) were selected for testing in combination for antagonist studies with the anti-LAG-3 monoclonal antibody.
- the EC50 of the anti-LAG-3 monoclonal antibody decreased from 690 pM (anti-LAG-3 only) to 40 pM (+133 pM anti-PD-1) or 200 pM (+13.3 pM anti-PD-1), which was a 17-fold and 3-fold increase in potency, respectively.
- the anti-LAG-3 antibody APE05505 was titrated in a dose response in the human CD4+ T-cell MLR assay described above. Based on the results from titrating the anti-LAG-3 antibody in multiple MLR assays, 2 nM (approximate EC50) and 0.2 nM (approximate EC10) were selected for testing in combination for antagonist studies with the anti-TIM-3 monoclonal antibody.
- the EC50 of the anti-LAG-3 mAb decreased from 1InM (anti-LAG-3 only) to 6 nM (+0.2 nM anti-TIM-3) or 3 nM (+2 nM anti-TIM-3), which was a 1.8-fold and 3.6-fold increase in potency, respectively.
- an inventive anti-LAG-3 monoclonal antibody can inhibit LAG-3 signaling and enhance T-cell activation in vivo in combination with an anti-PD-1 antibody.
- an anti-mouse LAG-3 surrogate monoclonal antibody (mAb C9B7W, BioXcell, West Riverside, N.H.) was tested alone or in combination with an anti-mouse PD-1 surrogate monoclonal antibody (mAb RMP1-14, BioXcell, West Riverside, N.H.) in the MC38 syngeneic tumor model.
- Groups of ten animals were injected subcutaneously with 1 ⁇ 10 6 MC38 cells. Ten days after inoculation, animals were randomized for tumor size. Mice were treated with 5 mg/kg of anti-PD-1 monoclonal antibody and/or 10 mg/kg or anti-LAG-3 monoclonal antibody on days 1, 4, 8, and 11, totaling four doses of each antibody or combination of antibodies.
- Tumors were measured twice weekly to assess response to treatment.
- the anti-PD-1+anti-LAG-3 combination was more efficacious in reducing tumor growth than each single agent alone.
- Complete response was observed in all ten animals of the group treated with the combination, as compared to seven animals in the PD-1-only group and no animals in the anti-LAG-3-only group.
- Nine animals showing a complete response from the combination group were then rechallenged by subcutaneous innoculation with 4 ⁇ 10 6 MC38 cells. None of the animals in the rechallenged group developed measurable tumor, while all control naive mice injected with the same amount of cells grew palpable tumor.
- the activities of the surrogate monoclonal antibodies described above also were tested alone or in combination in the Colon26 syngeneic tumor model.
- Groups of 12 animals were injected subcutaneously with 5 ⁇ 10 5 Colon26 cells. Mice were treated with 10 mg/kg of anti-PD-1 antibody and/or 10 mg/kg of anti-LAG-3 antibody on days 4, 7, 11, and 14, totaling four doses of each antibody or combination of antibodies. Tumors were measured twice weekly to assess response to treatment.
- the anti-PD-1+anti-LAG-3 combination was more efficacious for tumor growth than each single agent alone. Complete response was observed in 10 out of 12 animals in the combination group, as compared to three animals in the PD-1-only group and one animal in the anti-LAG-3-only group.
- This example demonstrates the effect of antibody isotype on anti-tumor activity of an anti-LAG-3 antibody alone or in combination with an anti-PD-1 antibody in a syngeneic mouse tumor model.
- mice LAG-3 of IgG1 (D265A) and IgG2a isotypes were created after sequencing and cloning the variable regions of an anti-mouse LAG-3 neutralizing antibody (mAb C9B7W, BioXcell, West Riverside, N.H.) from a rat hybridoma cell line and cloning into a mouse IgG1 or mouse IgG2a expression vector. These antibodies were then tested for efficacy both alone and in combination with a mouse IgG (D265A) surrogate antibody recognizing mouse PD-1 similarly created from a purchased rat antibody from BioXcel (mAb RMP1-14, West Riverside, N.H.).
- Colon26 colon adenocarcinoma cells (5 ⁇ 10 5 s.c.) were implanted into Balb/c mice and grown for 3 days. Mice were randomized into seven groups of 12 animals/group and dosed with each antibody or antibody combination on days 4, 7, 11, and 14 as set forth in Table 1. Mice injected with matched isotype antibodies served as a control. Tumor volumes were measured twice weekly until the end of the study.
- FIGS. 1A and 1B show that a single-agent anti-mouse LAG-3 antibody with minimal effector function (i.e., IgG1 (D265A)) has anti-tumor efficacy as compared with an anti-mouse LAG-3 antibody with effector function (i.e., IgG2a), which has no apparent effect on tumor growth.
- IgG1 D265A
- IgG2a anti-mouse LAG-3 antibody with effector function
- FIG. 1A shows an anti-mouse LAG-3 antibody with minimal effector function (i.e., IgG1(D265A)) in combination with a regimen of an anti-mouse PD-1 IgG1(D265A) antibody exhibited increased anti-tumor activity compared with the anti-mouse PD-1 IgG1(D265A) antibody alone.
- IgG1(D265A) an anti-mouse LAG-3 antibody with minimal effector function
- an anti-mouse LAG-3 antibody with in-tact effector function (IgG2a) in combination with an anti-mouse PD-1 antibody was less efficacious than anti-mouse PD-1 IgG1 (D265A) alone, suggesting that the effector function of the antibody possibly interfered with anti-mouse PD-1 mediated efficacy.
- FIG. 1B provides graphs of tumor volume over time for individual animals from treatment group 3 (anti-mouse PD-1 IgG1(D265A) antibody treated animals), group 7 (combination of anti-mouse PD-1 IgG(D265A) antibody with anti-mouse LAG-3 IgG(D265A) antibody), and group 6 (combination of anti-mouse PD-1 IgG1(D265A) antibody with anti-mouse LAG-3 IgG2 antibody).
- group 7 anti-mouse PD-1 IgG1(D265A) antibody with anti-mouse LAG-3 IgG1(D265A)
- 8/12 animals had no visible tumor growth by the end of the study.
- results of this example demonstrate that anti-mouse LAG-3 and anti-mouse PD-1 antibodies without effector function, alone and in combination, can inhibit tumor growth in a mouse syngeneic tumor model. Efficacy was not observed using an anti-mouse LAG-3 antibody with effector function and furthermore may interfere with anti-PD-1 mediated efficacy.
- This example demonstrates that an inventive anti-LAG-3 monoclonal antibody inhibitory activity can be differentiated from that of an anti-PD-1 monoclonal antibody in a mixed lymphocyte reaction based upon time of harvest and correlates with PD-1 and LAG-3 expression.
- a functional LAG-3 antagonist antibody was tested in a human CD4+ T-cell mixed lymphocyte reaction (MLR) assay in which activation of CD4+ T-cells in the presence of anti-LAG-3 antibodies is assessed by measuring IL-2 secretion.
- the anti-LAG-3 antibody was tested side by side with an antagonistic anti-PD-1 antibody, wherein the antibodies were added and/or harvested at different timepoints.
- isolated peripheral blood monocytes from a human donor were differentiated into dendritic cells (DCs) and then mixed with CD4+ T-cells isolated from a second donor.
- Inhibitory antibodies were added either at the start of the co-culture or 24 hours after the start of the co-culture.
- IL-2 levels were measured at 24 and 48 hours after antibody addition.
- Antagonism of LAG-3 and PD-1 was expected to result in increased T-cell activation as measured by increased IL-2 production.
- the anti-PD-1 antibody increased IL-2 secretion at both 24 and 48 hours post antibody addition, while the anti-LAG-3 antibody increased IL-2 secretion when measured at 48 hours in the MLR assay, but not at 24 hours.
- inhibitory anti-LAG-3 or anti-PD-1 antibodies were added at 24 hours after starting the co-culture and harvested at 72 hours, both antibodies were active and the EC50 appeared to be equivalent ( FIG. 2A ). This correlates with expression as increased PD-1 expression is observed at 24-72 hours, while LAG-3 appears to be expressed later in the assay at 48 and 72 hours ( FIG. 2B ).
- results of this example demonstrate that the effects of LAG-3 inhibition correlates with target expression, and that LAG-3 expression occurs temporally later than PD-1.
Abstract
Description
- Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 182,600 Byte ASCII (Text) file named “723163_ST25.TXT,” created on Feb. 2, 2016.
- Lymphocyte Activation Gene-3 (LAG-3), which is also known as CD223, is a member of the immunoglobulin supergene family and is structurally and genetically related to CD4. LAG-3 is expressed on T-cells, B cells, natural killer (NK) cells and plasmacytoid dendritic cells (ADCs). Like CD4, LAG-3 has been demonstrated to interact with MHC Class 11 molecules (Baixeras et al., J. Exp. Med., 176: 327-337 (1992)), but binds at a distinct site (Huard et al., Proc. Natl. Acad. Sci. USA, 94(11): 5744-5749 (1997)). In, particular, for example, a LAG-3 immunoglobulin fusion protein (sLAG-3Ig) directly and specifically binds via LAG-3 to MHC class II on the cell surface (Huard et al., Eur. J. Immunol., 26:1180-1186 (1996)).
- LAG-3 is upregulated following T-cell activation, and modulates T-cell function as well as T-cell homeostasis (Sierra et al., Expert Opin. Ther. Targets, 15(1):91-101 (2011)). The LAG-3/MHC class 11 interaction may play a role in down-regulating antigen-dependent stimulation of CD4+ T lymphocytes, as demonstrated in in vitro studies of antigen-specific T-cell responses in which the addition of anti-LAG-3 antibodies led to increased T-cell proliferation, higher expression of activation antigens such as CD25, and higher concentrations of cytokines such as interferon-gamma and interleukin-4 (Huard et al., Eur. J. Immunol., 24: 3216-3221 (1994)). CD4+CD25+ regulatory T-cells (Treg) also have been shown to express LAG-3 upon activation and antibodies to LAG-3 inhibit suppression by induced Treg cells, both in vitro and in vivo, suggesting that LAG-3 contributes to the suppressor activity of Treg cells (Huang et al. Immunity, 21: 503-513 (2004)). Furthermore, LAG-3 has been shown to negatively regulate T-cell homeostasis by regulatory T-cells in both T-cell-dependent and independent mechanisms (Workman, C. J. and Vignali, D. A., J. Immunol., 174: 688-695 (2005)).
- Subsets of conventional T-cells that are anergic or display impaired functions express LAG-3, and LAG-3+ T-cells are enriched at tumor sites and during chronic viral infections. However, while LAG-3 knockout mice have been shown to mount normal virus-specific CD4+ and CD8+ T-cell responses, suggesting a non-essential role for LAG-3, blockade of the PD-1/PD-L1 pathway combined with LAG-3 blockade improved viral control as compared with PD-L1 blockade alone (Blackburn et al., Nat. Immunol., 10: 29-37 (2009); and Richter et al., Int. Immunol., 22: 13-2 (2010)).
- In a self-tolerance/tumor mouse model where transgenic CD8+ T-cells were rendered unresponsive/anergic, in viva, LAG-3 blockade or deficiency in CD8+ T-cells enhanced T-cell proliferation, T-cell recruitment and effector functions, at the tumor site (Grosso et at, J. Clin. Invest., 117: 3383-92 (2007)).
- Inhibition of LAG-3 activity, such as through use of monoclonal antibodies, is currently under investigation as a therapeutic approach to treat viral infections and melanoma based on preclinical studies. For example, addition of soluble huLAG-3 fused to an Fc region enhanced the proliferation of antigen-specific T-cells to viral and tumor antigens, such as influenza matrix protein or melanoma antigen recognized by T-cells (MART-1), in PBMCs of healthy or cancer patients (Casati et al., J. Immunol, 180: 3782-3788 (2008)).
- There is a need for additional antagonists of LAG-3 (e.g., an antibody) that binds LAG-3 with high affinity and effectively neutralizes LAG-3 activity. The invention provides such LAG-3-binding agents.
- The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 Asp Ser Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 181), wherein (a) Xaa1 is asparagine (Asn) or serine (Ser), (b) Xaa2 is lysine (Lys), tyrosine (Tyr), or asparagine (Asn), (c) Xaa3 is lysine (Lys) or glutamine (Gln), (d) Xaa4 is isoleucine (e) or methionine (Met), (e) Xaa5 is alanine (Ala) or proline (Pro), (f) Xaa6 is glutamic acid (Glu) or methionine (Met), (g) Xaa6 is glycine (Gly), asparagine (Asn), or aspartic acid (Asp), (h) Xaa8 is glutamic acid (Glu) or glutamine (Q), (i) Xaa9 is alanine (Ala) or serine (Ser), (j) Xaa10 is glutamine (Gln) or arginine (Arg), (k) Xaa11 is aspartic acid (Asp) or asparagine (Asn), and (1) Xaa12 is glutamine (Gln) or lysine (Lys).
- The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Gln Val Gln Leu Gln Gln Trp Gly Ala Xaa1 Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Xaa2 Cys Xaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly Tyr Tyr Trp Xaa6 Trp e Arg Pro Xaa7 Lys Gly Leu Glu Trp e Gly Glu e Asn His Ser Gly Xaa8 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn Gln Xaa9 Ser Leu Lys Leu Xaa10 Xaa11 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr Gly Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 35), wherein (a) Xaa1 is arginine (Arg) or glycine (Gly), (b) Xaa2 is threonine (Thr) or isoleucine (Ile), (c) Xaa3 is threonine (Thr) or alanine (Ala), (d) Xaa4 is serine (Ser) or phenylalanine (Phe), (e) Xaa5 is serine (Ser) or phenylalanine (Phe), (f) Xaa6 is serine (Ser) or isoleucine (Ile), (g) Xaa7 is glycine (Gly) or arginine (Arg), (h) Xaa8 is serine (Ser) or asparagine (Asn), (i) Xaa9 is phenylalanine (Phe) or leucine (Leu), (j) Xaa10 is asparagine (Asn) or serine (Ser), (k) Xaa11 is serine (Ser) or phenylalanine (Phe), (1) Xaa12 is alanine (Ala) or valine (Val), and (m) Xaa13 is aspartic acid (Asp) or asparagine (Asn).
- The invention further provides an isolated immunoglobulin heavy chain polypeptide comprising SEQ ID NO: 190 or 191.
- The invention provides an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Xaa1 Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Xaa2 Ser Gln Ser Leu Val His Ser Asp Xaa3 Xaa4 Thr Tyr Leu His Tip Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Xaa Xaa Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys e Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gln Ser Thr Xaa Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu He Lys Arg Thr (SEQ ID NO: 57), wherein (a) Xaa1 is valine (Val) or isoleucine (Ile), (b) Xaa2 is cysteine (Cys) or serine (Ser), (c) Xaa3 is glycine (Gly) or serine (Ser), (d) Xaa4 is asparagine (Asn) or aspartic acid (Asp), (e)Xaa5 is lysine (Lys), glycine (Gly), asparagine (Asn), serine (Ser), or leucine (Leu), (f) Xaa6 is valine (Val) or isoleucine (le), (g) Xaa7 is serine (Ser), alanine (Ala), or glycine (Gly), and (h) Xaa8 is histidine (His) or tyrosine (Tyr).
- The invention provides an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp e Ser Asn Tyr Leu Asn Trp Tyr Gin Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val (SEQ ID NO: 89), wherein (a) the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 is deleted or is Tyr-Asp-Ala-Ser-Asn, and (b) Xaa6 is threonine (Thr) or isoleucine (Ile).
- The invention also provides isolated immunoglobulin light chain polypeptide comprising SEQ ID NO: 196 or 197.
- In addition, the invention provides isolated or purified nucleic acid sequences encoding the foregoing immunoglobulin polypeptides, vectors comprising such nucleic acid sequences, LAG-3-binding agents comprising the foregoing immunoglobulin polypeptides, nucleic acid sequences encoding such LAG-3-binding agents, vectors comprising such nucleic acid sequences, isolated cells comprising such vectors, compositions comprising such LAG-3-binding agents or such vectors with a pharmaceutically acceptable carrier, and methods of treating cancer or infectious diseases in mammals by administering effective amounts of such compositions to mammals.
-
FIG. 1A is a graph of mean tumor volume over time in mice implanted with Colon26 colon adenocarcinoma cells and injected with the indicated antibodies. Each data plot in the figure refers to the indicated treatment group. -
FIG. 1B is a graph of tumor volume over time of individual animals in three treatment groups of mice implanted with Colon26 colon adenocarcinoma cells and injected with the indicated antibodies. Each data plot in the graphs refers to an individual animal in the treatment group. -
FIG. 2A depicts IL-2 secretion by CD4+ T-cells in a mixed lymphocyte reaction (MLR) assay at varying concentrations of Anti PD-1 or Anti-LAG-3 antibodies. -
FIG. 2B depicts LAG-3 and PD-1 expression on CD4+ T-cells prior to (naïve) or subsequent to (24, 48, and 72 hour) exposure to dendritic cells. - The invention provides an isolated immunoglobulin heavy chain polypeptide and/or an isolated immunoglobulin light chain polypeptide, or a fragment (e.g., antigen-binding fragment) thereof. The term “immunoglobulin” or “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. The polypeptide is “isolated” in that it is removed from its natural environment. In a preferred embodiment, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (
C H1,C H2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (κ) or lambda (λ), based upon the amino acid sequences of their constant domains. In a typical immunoglobulin, each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other. - The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the hypervariable or complementary determining regions (CDRs). There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the β sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).
- The framework regions are connected by three complementarity determining regions (CDRs). As discussed above, the three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding. The CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions. While the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular toxicity via interactions with effector molecules and cells.
- The isolated immunoglobulin heavy chain polypeptide and the isolated immunoglobulin light chain polypeptide of the invention desirably bind to the protein encoded by the Lymphocyte Activation Gene-3 (LAG-3) (also referred to herein as “LAG-3 protein”). As discussed above, LAG-3 is a 498 amino acid protein that negatively regulates T-cell function and homeostasis (Triebel et al., J. Exp. Med., 171(5): 1393-1405 (1990); and Triebel F., Trends Immunol., 24(12): 619-22 (2003)). LAG-3 is a member of the immunoglobulin supergene family and is structurally and genetically related to CD4. The intra-cytoplasmic region of LAG-3 has been shown to interact with a protein denoted LAP, which is thought to be a signal transduction molecule involved in the downregulation of the CD3/TCR activation pathway (Iouzalen et al., Eur. J. Immunol., 31: 2885-2891 (2001)). Furthermore, CD4+CD25+ regulatory T-cells (Treg) have been shown to express LAG-3 upon activation and antibodies to LAG-3 inhibit suppression by induced Treg cells, both in vitro and in vivo, suggesting that LAG-3 contributes to the suppressor activity of Treg cells (Huang et al., Immunity, 21: 503-513 (2004)). However, a recent study suggests that LAG-3 expression on CD4+ T-cells renders them more susceptible to suppression by Tregs, rather than making Tregs more suppressive (see Durham et al., PLoS ONE, 9(11): e109080 (2014)). In certain circumstances, LAG-3 also has been shown to have immunostimulatory effects (see, e.g., Prigent et al., Eur. J. Immunol., 29: 3867-3876 (1999)); El Mir and Triebel, J. Immunol., 164: 5583-5589 (2000)); and Casati et al., Cancer Res., 66: 4450-4460 (2006)). The inventive isolated immunoglobulin heavy chain polypeptide and the inventive isolated immunoglobulin light chain polypeptide can form an agent that binds to LAG-3 and another antigen, resulting in a “dual reactive” binding agent (e.g., a dual reactive antibody). For example, the agent can bind to LAG-3 and to another negative regulator of the immune system such as, for example, programmed death 1 (PD-1) and/or T-cell immunoglobulin domain and
mucin domain 3 protein (TIM-3). - Antibodies which bind to LAG-3, and components thereof, are known in the art (see, e.g., U.S. Patent Application Publication Nos. 2010/0233183, 2011/0150892, and 2014/0093511). Anti-LAG-3 antibodies also are commercially available from sources such as, for example, Abcam (Cambridge, Mass.), and R&D Systems, Inc. (Minneapolis, Minn.).
- The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises the amino acid sequence Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 Asp Ser Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 181), wherein (a) Xaa1 is asparagine (Asn) or serine (Ser), (b) Xaa2 is lysine (Lys), tyrosine (Tyr), or asparagine (Asn), (c) Xaa3 is lysine (Lys) or glutamine (Gln), (d) Xaa4 is isoleucine (Ile) or methionine (Met), (e) Xaa5 is alanine (Ala) or proline (Pro), (f) Xaa6 is glutamic acid (Glu) or methionine (Met), (g) Xaa6 is glycine (Gly), asparagine (Asn), or aspartic acid (Asp), (h) Xaa8 is glutamic acid (Glu) or glutamine (Q), (i) Xaa9 is alanine (Ala) or serine (Ser), (j) Xaa10 is glutamine (Gln) or arginine (Arg), (k) Xaa11 is aspartic acid (Asp) or asparagine (Asn), and (1) Xaa12 is glutamine (Gln) or lysine (Lys).
- In another aspect, the immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of the amino acid sequence Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr e His Trp Val Xaa3 Gin Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp e Asp Xaa5 Glu Asn Xaa6 Asp Ser Glu Tyr Xaa7 Ser Lys Phe Xaa8 Gly Arg Val Thr Ile Thr Val Asp Thr Ser Thr Xaa9 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1), wherein (a) Xaa1 is asparagine (Asn) or serine (Ser), (b) Xaa2 is lysine (Lys), tyrosine (Tyr), or asparagine (Asn), (c) Xaa3 is lysine (Lys) or glutamine (Gin), (d) Xaa4 is isoleucine (Ile) or methionine (Met), (e) Xaa5 is alanine (Ala) or proline (Pro), (f) Xaa6 is glycine (Gly), asparagine (Asn), or aspartic acid (Asp), (g) Xaa7 is alanine (Ala) or serine (Ser), (h) Xaa8 is glutamine (Gin) or arginine (Arg), and (i) Xaa9 is aspartic acid (Asp) or asparagine (Asn).
- In one embodiment, the isolated immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, or SEQ ID NO: 186.
- The invention also provides an immunoglobulin heavy chain polypeptide that comprises, consists of, or consists essentially of the amino acid sequence Gin Val Gin Leu Gin Gin Trp Gly Ala Xaa1 Leu Leu Lys Pro Ser Glu Thr Lu Ser Leu Xaa2 Cys Xaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly Tyr Tyr Trp Xaa6 Trp Ile Arg Gin Pro Pro Xaa7 Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Xaa8 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr e Ser Val Asp Thr Ser Lys Asn Gin Xaa9 Ser Leu Lys Leu Xaa10 Xaa11 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr Gly Asp Tyr Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 35), wherein (a) Xaa1 is arginine (Arg) or glycine (Gly), (b) Xaa2 is threonine (Thr) or isoleucine (Ile), (c) Xaa3 is threonine (Thr) or alanine (Ala), (d) Xaa4 is serine (Ser) or phenylalanine (Phe), (e) Xaa5 is serine (Ser) or phenylalanine (Phe), (f) Xaa6 is serine (Ser) or isoleucine (Ile), (g) Xaa7 is glycine (Gly) or arginine (Arg), (h) Xaa8 is serine (Ser) or asparagine (Asn), (i) Xaa9 is phenylalanine (Phe) or leucine (Leu), (j) Xaa10 is asparagine (Asn) or serine (Ser), (k) Xaa11 is serine (Ser) or phenylalanine (Phe), (1) Xaa12 is alanine (Ala) or valine (Val), and (m) Xaa13 is aspartic acid (Asp) or asparagine (Asn).
- In one embodiment, the isolated immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.
- In another embodiment, there is provided an isolated immunoglobulin heavy chain polypeptide which comprises SEQ ID NO: 190 or 191. Examples of such a polypeptide include those comprising any one of SEQ ID NOs: 192-195.
- When the inventive immunoglobulin heavy chain polypeptide consists essentially of an amino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 56, SEQ ID NOS: 182-186, or SEQ ID NOS: 190-195, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation). When the inventive immunoglobulin heavy chain polypeptide consists of an amino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 56, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin heavy chain polypeptide).
- The invention provides an isolated immunoglobulin heavy chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 1-56. Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences). BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).
- In another embodiment, the invention provides an immunoglobulin light chain polypeptide that comprises, consists of, or consists essentially of, an isolated immunoglobulin light chain polypeptide which comprises the amino acid sequence Asp Xaa1 Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gin Pro Ala Ser Ile Ser Cys Arg Xaa2 Ser Gln Ser Leu Val His Ser Asp Xaa3 Xaa4 Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gin Ser Pro Gin Leu Leu Ile Tyr Xaa Xaa Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gin Ser Thr Xaa Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr (SEQ ID NO: 57), wherein (a) Xaa1 is valine (Val) or isoleucine (e), (b) Xaa2 is cysteine (Cys) or serine (Ser), (c) Xaa3 is glycine (Gly) or serine (Ser), (d) Xaa4 is asparagine (Asn) or aspartic acid (Asp), (e)Xaa5 is lysine (Lys), glycine (Gly), asparagine (Asn), serine (Ser), or leucine (Leu), (f) Xaa6 is valine (Val) or isoleucine (Ile), (g) Xaa7 is serine (Ser), alanine (Ala), or glycine (Gly), and (h) Xaa8 is histidine (His) or tyrosine (Tyr).
- In one embodiment, the isolated immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of an amino acid sequence of any one of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61. SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64. SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67. SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72, SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75, SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78. SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81. SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84, SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87. SEQ ID NO: 88. SEQ ID NO: 187. SEQ ID NO: 188, or SEQ ID NO: 189.
- The invention provides an isolated immunoglobulin light chain polypeptide which comprises, consists essentially of, or consists of the amino acid sequence Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val (SEQ ID NO: 89), wherein (a) the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 is deleted or is Tyr-Asp-Ala-Ser-Asn, and (b) Xaa6 is threonine (Thr) or isoleucine (Ile).
- The inventive immunoglobulin light chain polypeptide can include or lack the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 at positions 49-53 of SEQ ID NO: 89 when Xaa6 is threonine (Thr) or isoleucine (Ile). When the inventive immunoglobulin light chain polypeptide comprises the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5, each of Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 can be any suitable amino acid residue. Preferably. Xaa1 is tyrosine (Tyr), Xaa2 is aspartic acid (Asp). Xaa3 is alanine (Ala), Xaa4 is serine (Ser), and Xaa5 is asparagine (Asn). A preferred amino acid sequence of an immunoglobulin light chain polypeptide which includes the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 comprises SEQ ID NO: 90. When the immunoglobulin light chain polypeptide lacks the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5, the immunoglobulin light chain polypeptide preferably comprises the amino acid sequence SEQ ID NO: 91 or SEQ ID NO: 92.
- In another embodiment, provided is an isolated immunoglobulin light chain polypeptide which comprises SEQ ID NO: 196 or 197. Examples of such a polypeptide include those comprising any one of SEQ ID NOs: 198-200.
- When the inventive immunoglobulin light chain polypeptide consists essentially of an amino acid sequence of any one of SEQ ID NO: 57-SEQ ID NO: 92, SEQ ID NOs: 187-189, or SEQ ID NOs: 196-200, additional components can be included in the polypeptide that do not materially affect the polypeptide (e.g., protein moieties such as biotin that facilitate purification or isolation). When the inventive immunoglobulin light chain polypeptide consists of an amino acid sequence of any one of SEQ ID NO: 57-SEQ ID NO: 92, the polypeptide does not comprise any additional components (i.e., components that are not endogenous to the inventive immunoglobulin light chain polypeptide).
- The invention provides an isolated immunoglobulin light chain polypeptide which comprises an amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 57-SEQ ID NO: 92. Nucleic acid or amino acid sequence “identity” can be determined using the methods described herein.
- One or more amino acids of the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides can be replaced or substituted with a different amino acid. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
- Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).
- Aliphatic amino acids may be sub-divided into four sub-groups. The “large aliphatic non-polar sub-group” consists of valine, leucine, and isoleucine. The “aliphatic slightly-polar sub-group” consists of methionine, serine, threonine, and cysteine. The “aliphatic polar/charged sub-group” consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. The “small-residue sub-group” consists of glycine and alanine. The group of charged/polar amino acids may be sub-divided into three sub-groups: the “positively-charged sub-group” consisting of lysine and arginine, the “negatively-charged sub-group” consisting of glutamic acid and aspartic acid, and the “polar sub-group” consisting of asparagine and glutamine.
- Aromatic amino acids may be sub-divided into two sub-groups: the “nitrogen ring sub-group” consisting of histidine and tryptophan and the “phenyl sub-group” consisting of phenylalanine and tyrosine.
- The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
- Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free —OH can be maintained, and glutamine for asparagine such that a free —NH2 can be maintained.
- “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
- In addition, one or more amino acids can be inserted into the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides. Any number of any suitable amino acids can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide. In this respect, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids), can be inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide. Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted into the amino acid sequence of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide. In this respect, the amino acid(s) can be inserted into any one of the aforementioned immunoglobulin heavy chain polypeptides and/or light chain polypeptides in any suitable location. Preferably, the amino acid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of the immunoglobulin heavy chain polypeptide and/or light chain polypeptide.
- The inventive isolated immunoglobulin heavy chain polypeptide and light chain polypeptides are not limited to polypeptides comprising the specific amino acid sequences described herein. Indeed, the immunoglobulin heavy chain polypeptide or light chain polypeptide can be any heavy chain polypeptide or light chain polypeptide that competes with the inventive immunoglobulin heavy chain polypeptide or light chain polypeptide for binding to LAG-3. In this respect, for example, the immunoglobulin heavy chain polypeptide or light chain polypeptide can be any heavy chain polypeptide or light chain polypeptide that binds to the same epitope of LAG-3 recognized by the heavy and light chain polypeptides described herein. Antibody competition can be assayed using routine peptide competition assays which utilize ELISA, Western blot, or immunohistochemistry methods (see, e.g., U.S. Pat. Nos. 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4: 12 (2006)).
- The invention provides an isolated LAG-3-binding agent comprising, consisting essentially of, or consisting of one or more of the inventive isolated amino acid sequences described herein. By “LAG-3-binding agent” is meant a molecule, preferably a proteinaceous molecule, which binds specifically to the LAG-3 protein. Preferably, the LAG-3-binding agent is an antibody or a fragment (e.g., immunogenic fragment) thereof. The LAG-3-binding agent of the invention comprises, consists essentially of, or consists of the inventive isolated immunoglobulin heavy chain polypeptide and/or the inventive isolated immunoglobulin light chain polypeptide. In one embodiment, the LAG-3-binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide or the inventive immunoglobulin light chain polypeptide. In another embodiment, the LAG-3-binding agent comprises, consists essentially of, or consists of the inventive immunoglobulin heavy chain polypeptide and the inventive immunoglobulin light chain polypeptide.
- Any amino acid residue of the inventive immunoglobulin heavy chain polypeptide and/or the inventive immunoglobulin light chain polypeptide can be replaced, in any combination, with a different amino acid residue, or can be deleted or inserted, so long as the biological activity of the LAG-3-binding agent is enhanced or improved as a result of the amino acid replacements, insertions, and/or deletions. The “biological activity” of an LAG-3-binding agent refers to, for example, binding affinity for a particular LAG-3 epitope, neutralization or inhibition of LAG-3 binding to its receptor(s), neutralization or inhibition of LAG-3 activity in vivo (e.g., IC50), pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the LAG-3 protein, or with other proteins or tissues). Other biological properties or characteristics of an antigen-binding agent recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, and formulation. The aforementioned properties or characteristics can be observed, measured, and/or assessed using standard techniques including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or KINEXA™, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and/or secretion assays, and signal transduction and immunohistochemistry assays.
- The terms “inhibit” or “neutralize,” as used herein with respect to the activity of a LAG-3-binding agent, refer to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the biological activity of LAG-3, or a disease or condition associated with LAG-3. The isolated LAG-3-binding agent of the invention preferably inhibits or neutralizes the activity of LAG-3 by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the foregoing values.
- The isolated LAG-3-binding agent of the invention can be a whole antibody, as described herein, or an antibody fragment. The terms “fragment of an antibody,” “antibody fragment,” and “functional fragment of an antibody” are used interchangeably herein to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). The isolated LAG-3-binding agent can contain any LAG-3-binding antibody fragment. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab′ fragment, which results from breaking the disulfide bridge of an F(ab′)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
- In embodiments where the isolated LAG-3-binding agent comprises a fragment of the immunoglobulin heavy chain or light chain polypeptide, the fragment can be of any size so long as the fragment binds to, and preferably inhibits the activity of, LAG-3. In this respect, a fragment of the immunoglobulin heavy chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids. Similarly, a fragment of the immunoglobulin light chain polypeptide desirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values) amino acids.
- When the LAG-3-binding agent is an antibody or antibody fragment, the antibody or antibody fragment desirably comprises a heavy chain constant region (Fe) of any suitable class. Preferably, the antibody or antibody fragment comprises a heavy chain constant region that is based upon wild-type IgG1, IgG2, or IgG4 antibodies, or variants thereof. In some embodiments, the LAG-3 binding agent comprises an Fc region engineered to reduce or eliminate effector functions of the antibody. Engineered Fc regions with reduced or abrogated effector function are known in the art and commercially available, as are techniques for engineering Fc regions to reduce or eliminate effector function, any of which can be used in conjunction with the invention.
- The LAG-3-binding agent also can be a single chain antibody fragment. Examples of single chain antibody fragments include, but are not limited to, (i) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (ii) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009/0093024 A1.
- The isolated LAG-3-binding agent also can be an intrabody or fragment thereof. An intrabody is an antibody which is expressed and which functions intracellularly. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated VH and VL domains and scFvs. An intrabody can include sub-cellular trafficking signals attached to the N or C terminus of the intrabody to allow expression at high concentrations in the sub-cellular compartments where a target protein is located. Upon interaction with a target gene, an intrabody modulates target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment. Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in protein-protein, protein-DNA, or protein-RNA interactions.
- The isolated LAG-3-binding agent also can be an antibody conjugate. In this respect, the isolated LAG-3-binding agent can be a conjugate of (1) an antibody, an alternative scaffold, or fragments thereof, and (2) a protein or non-protein moiety comprising the LAG-3-binding agent. For example, the LAG-3-binding agent can be all or part of an antibody conjugated to a peptide, a fluorescent molecule, or a chemotherapeutic agent.
- The isolated LAG-3-binding agent can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody. By “chimeric” is meant an antibody or fragment thereof comprising both human and non-human regions. Preferably, the isolated LAG-3-binding agent is a humanized antibody. A “humanized” antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody. In one embodiment of the invention, CDRH3 of the inventive LAG-3-binding agent is obtained or derived from a mouse monoclonal antibody, while the remaining variable regions and constant region of the inventive LAG-3-binding agent are obtained or derived from a human monoclonal antibody.
- A human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described in, for example, Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). In certain embodiments, a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons. Inc., Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115-120 (2008)). In one embodiment, a humanized antibody can be produced using the methods described in, e.g., U.S. Patent Application Publication 2011/0287485 A1.
- In one embodiment, a CDR (e.g., CDR1, CDR2, or CDR3) or a variable region of the immunoglobulin heavy chain polypeptide and/or the immunoglobulin light chain polypeptide described herein can be transplanted (i.e., grafted) into another molecule, such as an antibody or non-antibody polypeptide, using either protein chemistry or recombinant DNA technology. In this regard, the invention provides an isolated LAG-3-binding agent comprising at least one CDR of an immunoglobulin heavy chain and/or light chain polypeptide as described herein. The isolated LAG-3-binding agent can comprise one, two, or three CDRs of an immunoglobulin heavy chain and/or light chain variable region as described herein.
- In a preferred embodiment, the LAG-3-binding agent binds an epitope of LAG-3 which blocks the binding of LAG-3 to MHC Class II molecules and inhibits LAG-3-mediated signaling. For example, the LAG-3 binding agent can bind to one or more of the four Ig-like extracellular domains (D1-D4) of the LAG-3 protein (see, e.g, Triebel et al., J. Exp. Med., 171(5): 1393-1405 (1990); and Bruniquel et al., Immunogenetics, 47: 96-98 (1997)). Preferably, the LAG-3 binding agent binds to domain 1 (D1) and/or domain (D2) of the LAG-3 protein. The invention also provides an isolated or purified epitope of LAG-3 which blocks the binding of LAG-3 to MHC Class II molecules in an indirect or allosteric manner.
- The invention also provides one or more isolated or purified nucleic acid sequences that encode the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and the inventive LAG-3-binding agent.
- The term “nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).
- The invention further provides a vector comprising one or more nucleic acid sequences encoding the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive LAG-3-binding agent. The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).
- In addition to the nucleic acid sequence encoding the inventive immunoglobulin heavy polypeptide, the inventive immunoglobulin light chain polypeptide, and/or the inventive LAG-3-binding agent, the vector preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, signal peptides (e.g., the osteonectin signal peptide), internal ribosome entry sites (IRES), and the like, that provide for the expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
- A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3′ or 5′ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.), LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123-144 (2005)).
- The term “enhancer” as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences.
- The vector also can comprise a “selectable marker gene.” The term “selectable marker gene,” as used herein, refers to a nucleic acid sequence that allow cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and described in, e.g., International Patent Application Publications WO 1992/008796 and WO 1994/028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Aced. Sci. USA, 78: 1527-1531(1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1-14 (1981); Santerre et al., Gene, 30: 147-156 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska & Szybalski, Proc. Natl. Aced. Sci. USA, 48: 2026-2034 (1962); Lowy et al., Cell, 22: 817-823 (1980); and U.S. Pat. Nos. 5,122,464 and 5,770,359.
- In some embodiments, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP.
- Other suitable vectors include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome. Such integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a site specific manner include, for example, components of the fip-in system from Invitrogen (Carlsbad, Calif.) (e.g.,
pcDNA™ 5/FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, Calif.). Examples of vectors that randomly integrate into host cell chromosomes include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Life Technologies (Carlsbad, Calif.), UCOE from Millipore (Billerica, Mass.), and pCI or pFN10A (ACT) FLEXI™ from Promega (Madison, Wis.). - Viral vectors also can be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentiviral-based pLP1 from Invitrogen (Carlsbad, Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.).
- Nucleic acid sequences encoding the inventive amino acid sequences can be provided to a cell on the same vector (i.e., in cis). A unidirectional promoter can be used to control expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control expression of multiple nucleic acid sequences. Nucleic acid sequences encoding the inventive amino acid sequences alternatively can be provided to the population of cells on separate vectors (i.e., in trans). Each of the nucleic acid sequences in each of the separate vectors can comprise the same or different expression control sequences. The separate vectors can be provided to cells simultaneously or sequentially.
- The vector(s) comprising the nucleic acid(s) encoding the inventive amino acid sequences can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby, including any suitable prokaryotic or eukaryotic cell. As such, the invention provides an isolated cell comprising the inventive vector. Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently.
- Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).
- Preferably, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.
- Suitable insect cells are described in, for example, Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4: 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).
- Preferably, mammalian cells are utilized in the invention. A number of suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO)(ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 42164220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Methods for selecting suitable mammalian host cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.
- In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin. Examples of human lymphoid cells lines include, without limitation, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85:1581-1585 (1988)), Raji cells (CCL-86), PER.C6 cells (Crucell Holland B.V., Leiden, The Netherlands), and derivatives thereof.
- A nucleic acid sequence encoding the inventive amino acid sequence may be introduced into a cell by “transfection,” “transformation,” or “transduction.” “Transfection,” “transformation.” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
- The invention provides a composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the inventive amino acid sequences, antigen-binding agent, or vector. Any suitable carrier can be used within the context of the invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).
- The invention further provides a method of treating a disorder in a mammal that is responsive to LAG-3 inhibition or neutralization. The method comprises administering the aforementioned composition to a mammal having a disorder that is responsive to LAG-3 inhibition or neutralization, whereupon the disorder is treated in the mammal. A disorder that is “responsive to LAG-3 inhibition” or “responsive to LAG-3 neutralization” refers to any disease or disorder in which a decrease in LAG-3 levels or activity has a therapeutic benefit in mammals, preferably humans, or the improper expression (e.g., overexpression) or increased activity of LAG-3 causes or contributes to the pathological effects of the disease or disorder. Disorders that are responsive to LAG-3 inhibition include, for example, cancer and infectious diseases. The inventive method can be used to treat any type of cancer known in the art, such as, for example, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma (see, e.g., Bhatia et al., Curr. Oncol. Rep., 13(6): 488-497 (2011)). The inventive method can be used to treat any type of infectious disease (i.e., a disease or disorder caused by a bacterium, a virus, a fungus, or a parasite). Examples of infectious diseases that can be treated by the inventive method include, but are not limited to, diseases caused by a human immunodeficiency virus (HIV), a respiratory syncytial virus (RSV), an influenza virus, a dengue virus, a hepatitis B virus (HBV, or a hepatitis C virus (HCV)). Administration of a composition comprising the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence induces an immune response against a cancer or infectious disease in a mammal. An “immune response” can entail, for example, antibody production and/or the activation of immune effector cells (e.g., T-cells).
- As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. To this end, the inventive method comprises administering a “therapeutically effective amount” of the LAG-3-binding agent. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the LAG-3-binding agent to elicit a desired response in the individual. For example, a therapeutically effective amount of a LAG-3-binding agent of the invention is an amount which decreases LAG-3 bioactivity in a human.
- Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the LAG-3-binding agent. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).
- A typical dose can be, for example, in the range of 1 μg/kg to 20 mg/kg of animal or human body weight; however, doses below or above this exemplary range are within the scope of the invention. The daily parenteral dose can be about 0.00001 μg/kg to about 20 mg/kg of total body weight (e.g., about 0.001 μg/kg, about 0.1 μg/kg, about 1 μg/kg, about 5 μg/kg, about 10 μg/kg, about 100 μg/kg, about 500 μg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two of the foregoing values), preferably from about 0.1 μg/kg to about 10 mg/kg of total body weight (e.g., about 0.5 μg/kg, about 1 μg/kg, about 50 μg/kg, about 150 μg/kg, about 300 μg/kg, about 750 μg/kg, about 1.5 mg/kg, about 5 mg/kg, or a range defined by any two of the foregoing values), more preferably from about 1 μg/kg to 5 mg/kg of total body weight (e.g., about 3 μg/kg, about 15 μg/kg, about 75 μg/kg, about 300 μg/kg, about 900 μg/kg, about 2 mg/kg, about 4 mg/kg, or a range defined by any two of the foregoing values), and even more preferably from about 0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg, about 13 mg/kg, or a range defined by any two of the foregoing values). Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
- The composition comprising an effective amount of the inventive immunoglobulin heavy chain polypeptide, the inventive immunoglobulin light chain polypeptide, the inventive LAG-3-binding agent, the inventive nucleic acid sequence encoding any of the foregoing, or the inventive vector comprising the inventive nucleic acid sequence can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition preferably is suitable for parenteral administration. The term “parenteral.” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
- Once administered to a mammal (e.g., a cross-reactive human), the biological activity of the inventive LAG-3-binding agent can be measured by any suitable method known in the art. For example, the biological activity can be assessed by determining the stability of a particular LAG-3-binding agent. In one embodiment of the invention, the LAG-3-binding agent (e.g., an antibody) has an in vivo half life between about 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the LAG-3-binding agent has an in vivo half life between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values). In another embodiment, the LAG-3-binding agent has an in vivo half life between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).
- The biological activity of a particular LAG-3-binding agent also can be assessed by determining its binding affinity to LAG-3 or an epitope thereof. The term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (KD). Affinity of a binding agent to a ligand, such as affinity of an antibody for an epitope, can be, for example, from about 1 picomolar (pM) to about 100 micromolar (μM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1 micromolar (μM), or from about 1 μM to about 100 μM). In one embodiment, the LAG-3-binding agent can bind to an LAG-3protein with a KD less than or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). In another embodiment, the LAG-3-binding agent can bind to LAG-3 with a KD less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). Immunoglobulin affinity for an antigen or epitope of interest can be measured using any art-recognized assay. Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXA™), antigen panning, and/or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 2001).
- The LAG-3-binding agent of the invention may be administered alone or in combination with other drugs (e.g., as an adjuvant). For example, the LAG-3-binding agent can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein. In this respect, the LAG-3-binding agent can be used in combination with at least one other anticancer agent including, for example, any chemotherapeutic agent known in the art, ionization radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer), and/or surgery. When the inventive method treats an infectious disease, the LAG-3-binding agent can be administered in combination with at least one anti-bacterial agent or at least one anti-viral agent. In this respect, the anti-bacterial agent can be any suitable antibiotic known in the art. The anti-viral agent can be any vaccine of any suitable type that specifically targets a particular virus (e.g., live-attenuated vaccines, subunit vaccines, recombinant vector vaccines, and small molecule anti-viral therapies (e.g., viral replication inhibitors and nucleoside analogs).
- In another embodiment, the inventive LAG-3 binding agent can be administered in combination with other agents that inhibit immune checkpoint pathways. For example, the inventive LAG-3 binding agent can be administered in combination with agents that inhibit or antagonize the programmed death 1 (PD-1), T-cell immunoglobulin domain and
mucin domain 3 protein (TIM-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways. Combination treatments that simultaneously target two or more of these immune checkpoint pathways have demonstrated improved and potentially synergistic antitumor activity (see, e.g., Sakuishi et al., J. Exp. Med., 207: 2187-2194 (2010); Ngiow et al., Cancer Res., 71: 3540-3551 (2011); and Woo et al., Cancer Res., 72: 917-927 (2012)). In one embodiment, the inventive LAG-3 binding agent is administered in combination with an antibody that binds to TIM-3 and/or an antibody that binds to PD-1. In this respect, the inventive method of treating a cancer or an infectious disease in a mammal can further comprise administering to the mammal a composition comprising (i) an antibody that binds to a TIM-3 protein and (ii) a pharmaceutically acceptable carrier or a composition comprising (i) an antibody that binds to a PD-1 protein and (ii) a pharmaceutically acceptable carrier. - In addition to therapeutic uses, the LAG-3-binding agent described herein can be used in diagnostic or research applications. In this respect, the LAG-3-binding agent can be used in a method to diagnose a disorder or disease in which the improper expression (e.g., overexpression) or increased activity of LAG-3 causes or contributes to the pathological effects of the disease or disorder. In a similar manner, the LAG-3-binding agent can be used in an assay to monitor LAG-3 protein levels in a subject being tested for a disease or disorder that is responsive to LAG-3 inhibition. Research applications include, for example, methods that utilize the LAG-3-binding agent and a label to detect an LAG-3 protein in a sample. e.g., in a human body fluid or in a cell or tissue extract. The LAG-3-binding agent can be used with or without modification, such as covalent or non-covalent labeling with a detectable moiety. For example, the detectable moiety can be a radioisotope (e.g., 3H, 14C, 32P, 35S, or 125I), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), or prosthetic groups. Any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed in the context of the invention (see, e.g., Hunter et al., Nature, 194: 495-496 (1962); David et al., Biochemistry, 13: 1014-1021(1974); Pain et al., J. Immunol. Meth., 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982)).
- LAG-3 protein levels can be measured using the inventive LAG-3-binding agent by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA), and FACS. Normal or standard expression values of LAG-3 can be established using any suitable technique, e.g., by combining a sample comprising, or suspected of comprising, LAG-3 with a LAG-3-specific antibody under conditions suitable to form an antigen-antibody complex. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amount of LAG-3 polypeptide expressed in a sample is then compared with a standard value.
- The LAG-3-binding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a diagnostic assay. If the LAG-3-binding agent is labeled with an enzyme, the kit desirably includes substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides a detectable chromophore or fluorophore). In addition, other additives may be included in the kit, such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. The reagents may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
- The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
- This example demonstrates a method of generating monoclonal antibodies directed against human LAG-3.
- The gene encoding the extracellular domain (ECD) of human LAG-3 was fused to either mouse IgG2a (human LAG-3 mIgG2a Fc) or a disabled form of wasabi fluorescent protein (dWFP human LAG-3) to produce antigen for use in mouse immunization and hybridoma screening. Specifically, female Swiss Webster (SWR) mice were purchased from Harlan Laboratories, Inc. (Indianapolis, Ind.) and divided into two groups. After six days of acclimatization, one group of animals was immunized with four to six doses of purified human LAG-3 mIgG2a Fc at 50 μg/mouse at intervals of three to four weeks using complete Freund's adjuvant (CFA) or incomplete Freunds adjuvant (IFA). The second group of animals was injected with four to six doses at intervals of three to four weeks alternating between human LAG-3 mIgG2a F or dWFP human LAG-3 ECD. CFA or IFA was also used as adjuvant in the second group. Animals were bled for measurement of the serum titer to human LAG-3 as assessed by binding to cell surface human LAG-3. CHO-S cells were transfected with a full length human LAG-3 extracellular domain fused to the H-2Kk transmembrane domain (CHO-S huLAG-3 ECD cells). Sera were diluted from 1:1,000-1:1,000,000 and incubated with the CHO-S huLAG-3 ECD cells for 30 minutes at 4° C. Cells were centrifuged, washed once with PBS/1% BSA, and incubated with PE-conjugated (Southern Biotech, Birmingham, Ala.) or ALEXAFLUOR™ 647- (Jackson Immunoresearch, West Grove, Pa.) labeled goat anti-mouse IgG (H+L) for 30 minutes at 4° C. Cells were washed twice in PBS/1% BSA, resuspended in PBS/I % BSA, and analyzed on a BD FACSARRAY™ Bioanalyzer (BD Biosciences, Franklin Lakes, N.J.). Based on titer readings, one animal from each group was boosted 3 days prior to spleen collection. Single cell suspensions were prepared from spleen tissue and used for generation of hybridomas by cell fusion using standard techniques. Two different myeloma cell lines were used for fusion, FO (described in de St. Groth and Scheidegger, J. Immunol. Methods, 35: 1-21 (1980)) and P3X63Ag8.653 (described in Kearney et al., J. Immunol., 123: 1548-1550 (1979)).
- Hybridoma supernatants were screened for binding to CHO-S huLAG-3 ECD cells and compared to binding to untransfected CHO-S cells as described above. Based upon binding CHO-S huLAG-3 ECD cells, hybridomas were transferred to 48-well plates and expanded.
- Supematants were then tested for the ability to block binding of human LAG-3 mIgG2a Fc labeled with DyL650 (human LAG-3 mIgG2a Fc DyL650) to Daudi cells, which is a B-cell line that endogenously expresses high levels of MHCII (the LAG-3 receptor). Briefly, human LAG-3 mIgG2a Fc DyL650 was pre-incubated with control IgG or anti-human LAG-3 candidate monoclonal antibodies prior to addition to Daudi cells. Blocking was measured by reduction in fluorescence to Daudi cells using a BD FACSARRAY™ Bioanalyzer. These hybridomas were then subcloned and expanded to plate for generation of exhaust supernatant. Antibodies were subsequently purified and retested to confirm both binding to CHO-S huLAG-3 ECD cells and blocking ability in the Daudi assay.
- The results of this example confirm the production of anti-LAG-3 monoclonal antibodies using hybridoma cell technology.
- This example describes the design and generation of CDR-grafted and chimeric anti-LAG-3 monoclonal antibodies.
- Antibodies from the hybridomas described in Example 1 were isotyped, subjected to RT-PCR for cloning the antibody heavy chain variable region (VH) and light chain variable region (VL), and sequenced. Specifically, RNA was isolated from cell pellets of hybridoma clones (1×106 cells/pellet) using the RNEASY™ kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using oligo-dT-primed SUPERSCRIPT™ III First-Strand Synthesis System (Life Technologies, Carlsbad, Calif.). PCR amplification of the VL utilized a pool of degenerate mouse VL forward primers (see Kontermann and Dubel, eds., Antibody Engineering, Springer-Verlag, Berlin (2001)) and a mouse a constant region reverse primer. PCR amplification of the VH utilized a pool of degenerate mouse VH forward primers (Kontermann and Dubel, supra) and a mouse γ1 or γ2a constant region reverse primer (based on isotyping of purified antibody from each clone) with the protocol recommended in the SUPERSCRIPT™ III First-Stand Synthesis System (Life Technologies, Carlsbad, Calif.). PCR products were purified and cloned into pcDNA3.3-TOPO (Life Technologies, Carlsbad, Calif.).
- Individual colonies from each cell pellet were selected and sequenced using standard Sanger sequencing methodology (Genewiz, Inc., South Plainfield, N.J.). Variable region sequences were examined and aligned with the closest human heavy chain or light chain V-region germline sequence. Three antibodies were selected for CDR-grafting, which were denoted (1) 5.B11, (2) 5.D7, and (3) 1.E10.
- CDR-grafted antibody sequences were designed by cloning CDR residues from each of the above-described mouse antibodies into the closest human germline homolog. CDR-grafted antibody variable regions were synthesized and expressed with human IgG/κ constant regions for analysis. In addition, mouse:human chimeric antibodies were constructed using the variable regions of the above-described mouse antibodies linked to human IgG/κ constant regions. Chimeric and CDR-grafted antibodies were characterized for binding to CHO-S huLAG-3 ECD cells and for activity in the human LAG-3 ECD/Daudi blocking assay as described above.
- The functional antagonist activity of chimeric and CDR-grafted antibodies also was tested in a human CD4⋅ T-cell:dendritic cell mixed lymphocyte reaction (MLR) assay in which activation of CD4⋅ T-cells in the presence of anti-LAG-3 antibodies is assessed by measuring IL-2 secretion. Because LAG-3 is a negative regulator of T-cell function, antagonism of LAG-3 was expected to result in increased T-cell activation as measured by increased IL-2 production. The 5.B11, 5.D7, and 1.E10 CDR-grafted antibodies demonstrated antagonistic activity in the MLR assay as measured by an increase in IL-2 activity.
- The results of this example demonstrate a method of generating chimeric and CDR-grafted monoclonal antibodies that specifically bind to and inhibit LAG-3.
- This example demonstrates affinity maturation of humanized monoclonal antibodies directed against human LAG-3.
- CDR-grafted antibodies derived from two of the original murine monoclonal antibodies described in Example 2, 5.D7 and 1.E10, were subjected to affinity maturation via in silico somatic hypermutation (iSHM). This method incorporates mutations as predicted by computational analysis comparing in vivo matured antibody sequences, as downloaded from NCBI, and comparing them to germline human IGHV, IGKV, and IGLV sequences and their allelic forms (as described in Bowers et al., J. Biol. Chem., 288(11):7688-7696 (2013)). The LAG-3 binding properties of resultant antibodies were assayed using surface plasmon resonance (SPR) as well as ability to bind to CHO-S huLAG-3 ECD cells as described previously. Solution-based affinity analyses were also performed on using a
KINEXA™ 3000 assay (Sapidyne Instruments, Boise, Id.), and results were analyzed using KINEXA™ Pro Software 3.2.6. Experimental parameters were selected to reach a maximum signal with antibody alone between 0.8 and 1.2 V, while limiting nonspecific binding signal with buffer alone to less than 10% of the maximum signal. Azlactone beads (50 mg) were coated with antigen by diluting in a solution of human or cynoWFP-LAG-3 (50 μg/mL in 1 mL) in 50 mM Na2CO3. The solution was rotated at room temperature for 2 hours, and beads were pelleted in a picofuge and washed twice with blocking solution (10 mg/mL BSA, 1 M Tris-HCl, pH 8.0). Beads were resuspended in blocking solution (1 mL), rotated at room temperature for 1 hour, and diluted in 25 volumes PBS/0.02% NaN3. For affinity measurement, the secondary antibody was ALEXFLUOR™ 647 dye-anti-human IgG (500 ng/mL). Sample antibody concentrations were held constant (50 pM or 75 pM), while human or cynomolgus WFP-LAG-3 antigen was titrated using a three-fold dilutions series from 1 μM to 17 pM. All samples were diluted in PBS, 0.2% NaN3, 1 mg/mL BSA and allowed to equilibrate at room temperature for 30 hours. Additionally, samples containing only antibody and only buffer were tested in order to determine maximum signal and nonspecific binding signal, respectively. - Thermal stability of the selected antibodies was assessed using a Thermofluor assay as described in McConnell et al., Protein Eng. Des. Sel., 26: 151 (2013). This assay assesses stability through the ability of a hydrophobic fluorescent dye to bind to hydrophobic patches on the protein surface which are exposed as the protein unfolds. The temperature at which 50% of the protein unfolds (Tm) is determined to measure thermal stability. This assay demonstrated that 5.D7 monoclonal antibody variants had acceptable melting temperatures (Tms) (i.e., greater than 70° C.) that were suitable for drug development.
- De-risking of potential issues related to in vivo pharmokinetics of the tested antibodies was undertaken through assessment of non-specific binding to target negative cells (see, e.g., Hotzel et al., mAbs, 4: 753-760 (2012)). Antibodies were tested for binding to HEK 293f cells using a flow cytometry-based assay. The results indicated that non-specific binding was low for 5.D7 and could be further eliminated through second step purification.
- The results of this example confirm a method of affinity maturing humanized monoclonal antibodies directed against LAG-3.
- This example demonstrates a method of identifying antibodies directed against human LAG-3 from an evolvable library.
- An IgG evolvable library, based on germline sequence V-gene segments joined to human donor-derived recombined (D)J regions, was constructed as described in Bowers et al. Proc. Natl. Acad Sci. USA, 108(51): 20455-20460 (2011). IgG heavy chain (HC) and light chain (LC) were cloned into separate episomal vectors (Horlick et al., Gene, 243(1-2): 187-194 (2000)), with each vector encoding a distinct antibiotic selectable marker. The HC vector was formatted such that antibody was presented both on the cell surface as well as secreted into the tissue culture medium (Horlick et al., J. Biol. Chem., 288(27): 19861-19869 (2013)). The diverse sets of HCs and LCs were co-transfected into HEK293 cells and expanded to approximately 10 cells. The cell library was then subjected to two rounds each of negative selection against streptavidin (SA)-coupled magnetic beads alone (catalog #11047, Life Technologies, Carlsbad, Calif.) and irrelevant biotinylated antigen coated with SA-coupled magnetic beads. One round of positive selection was then performed using either magnetic beads coated directly with human LAG-3 mIgG2a Fc or with SA-coupled magnetic beads coated with biotinylated LAG-3 ECD mIgG1 Fc. The positively selected cells were diluted and plated in 96-well format at an approximate density of 1-10 cells/well. Resulting colonies were expanded into daughter plates and a portion of each population was tested for binding to LAG-3 ECD mIgG1 Fc DyL650 by FACSARRAY™ analysis. Antibodies secreted into the supernatant also were tested by BIACORE™ for ability to bind to LAG-3 ECD mIgG1 Fc.
- Cells that showed specific staining to human LAG-3 mIgG2a Fc DyL650 by FACSARRAY™ analysis and/or binding by BIACORE™ were expanded for sorting and submitted for sequencing to recover the specific HC/LC combinations capable of binding to human LAG-3. The open reading frames (ORFs) encoding the HCs and LCs of the antibodies found in the cell populations were rescued by PCR. Generally, multiple HC/LC sequences were found by sequencing. In some cases the desired HC/LC combinations were identified by enriching cells expressing monoclonal antibodies of interest by first FACS sorting with human LAG-3 mIgG2a Fc DyL650. Populations of cells exhibiting high antibody expression and positive for binding to human LAG-3 mIgG2a Fc DyL650 were isolated and subjected to subsequent sequence analysis. Overall, 12 different HC/LC pairs were identified as potential specific anti-LAG-3 antibody hits suitable for further characterization. These strategies were labeled A1/A14, A2, A3/A17, A4/A19, A5/A16, A6, A8/A20, A9, A10/A15, A11, A12, and A13.
- Antibodies also were characterized for their ability to bind to cynomolgus monkey LAG-3 protein (cyno LAG-3). As these germline antibodies identified from the library were too weak to bind to antigen expressed on the cell surface, soluble antigen similar to the human antigen was labeled with DyL650 (cyno LAG-3 mIgG2a Fc DyL650) and then incubated with HEK293 cells displaying antibody strategies on the cell surface. Eight antibody strategies identified from the evolvable library were tested and demonstrated an ability to bind to cyno LAG-3 ECD mIgG1 Fc.
- The results of this example confirm that monoclonal antibodies directed against human and non-human LAG-3 can be identified using an evolvable library.
- This example demonstrates affinity maturation of antibodies directed against human LAG-3 identified using an evolvable library.
- Stable cell lines co-expressing the HC and LC of each antibody identified from the evolvable library described in Example 4 were transfected with activation induced cytidine deaminase (AID) to initiate in vitro SHM. AID was also transfected directly into the original mixed population of cells expanded from the library screen. In all cases, cell populations were stained for both IgG expression and binding to antigen, collected by flow cytometry as a bulk population, and then expanded for sequence analysis by next generation sequencing (NGS). This process was repeated iteratively to accumulate SHM-derived mutations in the variable regions of both the heavy and light chains, and their derivatives, for each strategy. Improvements in affinity were monitored by (1) SPR, (2) ability to bind to CHO-S huLAG-3 ECD cells, and (3) activity in the MLR assay. As the affinity of each antibody improved, the stringency of selection was increased until affinity goals were achieved through the identification and recombination of novel mutations.
- Thermal stability of the selected antibodies was assessed using a Thermofluor assay as described above. This assay demonstrated that select monoclonal antibodies from the A17 strategy had acceptable Tms that were suitable for drug development. Antibodies also were tested for binding to HEK 293f cells using a flow cytometry-based assay. The results indicated that non-specific binding was low for select A17 candidates.
- Selected antibodies were tested for the ability to block binding of human LAG-3 mIgG2a Fc labeled with DyL650 (human LAG-3 mIgG2a Fc DyL650) to Daudi cells, as described above. A dose range of neutralizing antibodies was preincubated with the soluble LAG-3 and analyzed by flow cytometry. Certain affinity-matured anti-LAG-3 antibodies completely inhibited the interaction of soluble LAG-3 with MHCII.
- The results of this example confirm a method of affinity maturing monoclonal antibodies directed against LAG-3 identified using an evolvable library.
- This example demonstrates that an inventive anti-LAG-3 monoclonal antibody can inhibit LAG-3 signaling and enhance T-cell activation in vitro alone and in combination with an anti-PD-1 antibody or an anti-TIM-3 antibody.
- To establish parameters for anti-LAG-3 and anti-PD-1 combination studies, the anti-PD-1 antibody APE02058 was titrated in a dose-response in the human CD4+ T-cell MLR assay described above. Based on the results from titrating the anti-PD-1 antibody in multiple MLR assays, 133 pM (approximate EC50) and 13 pM (approximate EC10) were selected for testing in combination for antagonist studies with the anti-LAG-3 monoclonal antibody. In combination with 133 pM or 13.3 pM of anti PD-1, the EC50 of the anti-LAG-3 monoclonal antibody decreased from 690 pM (anti-LAG-3 only) to 40 pM (+133 pM anti-PD-1) or 200 pM (+13.3 pM anti-PD-1), which was a 17-fold and 3-fold increase in potency, respectively.
- To establish parameters for anti-LAG-3 and anti-TIM-3 combination studies, the anti-LAG-3 antibody APE05505 was titrated in a dose response in the human CD4+ T-cell MLR assay described above. Based on the results from titrating the anti-LAG-3 antibody in multiple MLR assays, 2 nM (approximate EC50) and 0.2 nM (approximate EC10) were selected for testing in combination for antagonist studies with the anti-TIM-3 monoclonal antibody. In combination with 2 nM or 0.2 nM of anti LAG-3, the EC50 of the anti-LAG-3 mAb decreased from 1InM (anti-LAG-3 only) to 6 nM (+0.2 nM anti-TIM-3) or 3 nM (+2 nM anti-TIM-3), which was a 1.8-fold and 3.6-fold increase in potency, respectively.
- The results of this example demonstrate that the inventive LAG-3 binding agent can inhibit LAG-3 biological activity alone and in combination with antagonists of other negative regulators of the immune system.
- This example demonstrates that an inventive anti-LAG-3 monoclonal antibody can inhibit LAG-3 signaling and enhance T-cell activation in vivo in combination with an anti-PD-1 antibody.
- The activity of an anti-mouse LAG-3 surrogate monoclonal antibody (mAb C9B7W, BioXcell, West Lebanon, N.H.) was tested alone or in combination with an anti-mouse PD-1 surrogate monoclonal antibody (mAb RMP1-14, BioXcell, West Lebanon, N.H.) in the MC38 syngeneic tumor model. Groups of ten animals were injected subcutaneously with 1×106 MC38 cells. Ten days after inoculation, animals were randomized for tumor size. Mice were treated with 5 mg/kg of anti-PD-1 monoclonal antibody and/or 10 mg/kg or anti-LAG-3 monoclonal antibody on
days - The activities of the surrogate monoclonal antibodies described above also were tested alone or in combination in the Colon26 syngeneic tumor model. Groups of 12 animals were injected subcutaneously with 5×105 Colon26 cells. Mice were treated with 10 mg/kg of anti-PD-1 antibody and/or 10 mg/kg of anti-LAG-3 antibody on
days 4, 7, 11, and 14, totaling four doses of each antibody or combination of antibodies. Tumors were measured twice weekly to assess response to treatment. The anti-PD-1+anti-LAG-3 combination was more efficacious for tumor growth than each single agent alone. Complete response was observed in 10 out of 12 animals in the combination group, as compared to three animals in the PD-1-only group and one animal in the anti-LAG-3-only group. Nine animals showing complete response from the combination group were then rechallenged with 5×105 Colon26 cells. None of the animals in the rechallenged group developed measurable tumor, while all the control naive mice injected with the same amount of cells grew palpable tumor. - The results of this example demonstrate that the inventive LAG-3 binding agent, in combination with antagonists of other negative regulators of the immune system, can inhibit LAG-3 biological activity in vivo.
- This example demonstrates the effect of antibody isotype on anti-tumor activity of an anti-LAG-3 antibody alone or in combination with an anti-PD-1 antibody in a syngeneic mouse tumor model.
- Surrogate antibodies recognizing mouse LAG-3 of IgG1 (D265A) and IgG2a isotypes were created after sequencing and cloning the variable regions of an anti-mouse LAG-3 neutralizing antibody (mAb C9B7W, BioXcell, West Lebanon, N.H.) from a rat hybridoma cell line and cloning into a mouse IgG1 or mouse IgG2a expression vector. These antibodies were then tested for efficacy both alone and in combination with a mouse IgG (D265A) surrogate antibody recognizing mouse PD-1 similarly created from a purchased rat antibody from BioXcel (mAb RMP1-14, West Lebanon, N.H.). Specifically, Colon26 colon adenocarcinoma cells (5×105 s.c.) were implanted into Balb/c mice and grown for 3 days. Mice were randomized into seven groups of 12 animals/group and dosed with each antibody or antibody combination on
days 4, 7, 11, and 14 as set forth in Table 1. Mice injected with matched isotype antibodies served as a control. Tumor volumes were measured twice weekly until the end of the study. -
TABLE 1 Group Treatment Dose 1 Isotype IgG2a + Isotype IgG1(D265A) 10 mg/kg, 1 mg/ kg 2 Isotype IgG1 (D265A) 10 mg/ kg 3 Anti-mPD-1 IgG1(D265A) 1 mg/ kg 4 Anti-mLAG-3 IgG2a 10 mg/ kg 5 Anti-mLAG-3 IgG1(D265A) 10 mg/kg 6 Anti-mPD-1 IgG1(D265A) + 1 mg/kg, 10 mg/kg Anti-mLAG-3 IgG2a 7 Anti-mPD-1 IgG1(D265A) + 1 mg/kg, 10 mg/kg Anti-mLAG-3 IgG1(D265A) - Results for this experiment are shown in
FIGS. 1A and 1B , which show that a single-agent anti-mouse LAG-3 antibody with minimal effector function (i.e., IgG1 (D265A)) has anti-tumor efficacy as compared with an anti-mouse LAG-3 antibody with effector function (i.e., IgG2a), which has no apparent effect on tumor growth. - In addition,
FIG. 1A shows an anti-mouse LAG-3 antibody with minimal effector function (i.e., IgG1(D265A)) in combination with a regimen of an anti-mouse PD-1 IgG1(D265A) antibody exhibited increased anti-tumor activity compared with the anti-mouse PD-1 IgG1(D265A) antibody alone. However, an anti-mouse LAG-3 antibody with in-tact effector function (IgG2a) in combination with an anti-mouse PD-1 antibody was less efficacious than anti-mouse PD-1 IgG1 (D265A) alone, suggesting that the effector function of the antibody possibly interfered with anti-mouse PD-1 mediated efficacy. -
FIG. 1B provides graphs of tumor volume over time for individual animals from treatment group 3 (anti-mouse PD-1 IgG1(D265A) antibody treated animals), group 7 (combination of anti-mouse PD-1 IgG(D265A) antibody with anti-mouse LAG-3 IgG(D265A) antibody), and group 6 (combination of anti-mouse PD-1 IgG1(D265A) antibody with anti-mouse LAG-3 IgG2 antibody). In group 7 (anti-mouse PD-1 IgG1(D265A) antibody with anti-mouse LAG-3 IgG1(D265A)), 8/12 animals had no visible tumor growth by the end of the study. By contrast, only 3/12 animals in group 6 (anti-mouse PD-1 IgG(D265A) antibody with anti-mouse LAG-3 IgG2 antibody) had no visible tumor by the end of the study. In group 3 (anti-mouse PD-1 IgG1 (D265A) alone), 6/12 animals were tumor free by the end of study, suggesting possible interference by the effector function of the anti-mouse LAG-3 IgG2 antibody when dosed in combination with the anti-mouse PD-1 IgG1 (D265A) antibody. - The results of this example demonstrate that anti-mouse LAG-3 and anti-mouse PD-1 antibodies without effector function, alone and in combination, can inhibit tumor growth in a mouse syngeneic tumor model. Efficacy was not observed using an anti-mouse LAG-3 antibody with effector function and furthermore may interfere with anti-PD-1 mediated efficacy.
- This example demonstrates that an inventive anti-LAG-3 monoclonal antibody inhibitory activity can be differentiated from that of an anti-PD-1 monoclonal antibody in a mixed lymphocyte reaction based upon time of harvest and correlates with PD-1 and LAG-3 expression.
- A functional LAG-3 antagonist antibody was tested in a human CD4+ T-cell mixed lymphocyte reaction (MLR) assay in which activation of CD4+ T-cells in the presence of anti-LAG-3 antibodies is assessed by measuring IL-2 secretion. The anti-LAG-3 antibody was tested side by side with an antagonistic anti-PD-1 antibody, wherein the antibodies were added and/or harvested at different timepoints. Specifically, isolated peripheral blood monocytes from a human donor were differentiated into dendritic cells (DCs) and then mixed with CD4+ T-cells isolated from a second donor. Inhibitory antibodies were added either at the start of the co-culture or 24 hours after the start of the co-culture. IL-2 levels were measured at 24 and 48 hours after antibody addition.
- Antagonism of LAG-3 and PD-1 was expected to result in increased T-cell activation as measured by increased IL-2 production. When added at the start of the assay, the anti-PD-1 antibody increased IL-2 secretion at both 24 and 48 hours post antibody addition, while the anti-LAG-3 antibody increased IL-2 secretion when measured at 48 hours in the MLR assay, but not at 24 hours. When inhibitory anti-LAG-3 or anti-PD-1 antibodies were added at 24 hours after starting the co-culture and harvested at 72 hours, both antibodies were active and the EC50 appeared to be equivalent (
FIG. 2A ). This correlates with expression as increased PD-1 expression is observed at 24-72 hours, while LAG-3 appears to be expressed later in the assay at 48 and 72 hours (FIG. 2B ). - The results of this example demonstrate that the effects of LAG-3 inhibition correlates with target expression, and that LAG-3 expression occurs temporally later than PD-1.
- All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
- The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
- Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Claims (18)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/074,269 US20210095026A1 (en) | 2015-02-03 | 2020-10-19 | Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562111486P | 2015-02-03 | 2015-02-03 | |
PCT/US2016/016424 WO2016126858A2 (en) | 2015-02-03 | 2016-02-03 | Antibodies directed against lymphocyte activation gene 3 (lag-3) |
US201715548405A | 2017-08-02 | 2017-08-02 | |
US17/074,269 US20210095026A1 (en) | 2015-02-03 | 2020-10-19 | Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/548,405 Continuation US10836824B2 (en) | 2015-02-03 | 2016-02-03 | Antibodies directed against lymphocyte activation gene 3 (LAG-3) |
PCT/US2016/016424 Continuation WO2016126858A2 (en) | 2015-02-03 | 2016-02-03 | Antibodies directed against lymphocyte activation gene 3 (lag-3) |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210095026A1 true US20210095026A1 (en) | 2021-04-01 |
Family
ID=56564868
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/548,405 Active 2037-01-03 US10836824B2 (en) | 2015-02-03 | 2016-02-03 | Antibodies directed against lymphocyte activation gene 3 (LAG-3) |
US16/400,198 Abandoned US20190256596A1 (en) | 2015-02-03 | 2019-05-01 | Antibodies directed against lymphocyte activation gene 3 (lag-3) |
US17/074,269 Pending US20210095026A1 (en) | 2015-02-03 | 2020-10-19 | Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/548,405 Active 2037-01-03 US10836824B2 (en) | 2015-02-03 | 2016-02-03 | Antibodies directed against lymphocyte activation gene 3 (LAG-3) |
US16/400,198 Abandoned US20190256596A1 (en) | 2015-02-03 | 2019-05-01 | Antibodies directed against lymphocyte activation gene 3 (lag-3) |
Country Status (15)
Country | Link |
---|---|
US (3) | US10836824B2 (en) |
EP (2) | EP3253798A4 (en) |
JP (1) | JP6949713B2 (en) |
KR (1) | KR20170116067A (en) |
CN (2) | CN114634573A (en) |
BR (1) | BR112017016638B1 (en) |
CA (1) | CA2975753A1 (en) |
EA (1) | EA201791742A1 (en) |
HK (2) | HK1246814A1 (en) |
IL (1) | IL253718B (en) |
MA (1) | MA41463A (en) |
MX (2) | MX2017009904A (en) |
SG (2) | SG11201706199RA (en) |
WO (1) | WO2016126858A2 (en) |
ZA (1) | ZA201705303B (en) |
Families Citing this family (74)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JO3663B1 (en) | 2014-08-19 | 2020-08-27 | Merck Sharp & Dohme | Anti-lag3 antibodies and antigen-binding fragments |
MA41463A (en) | 2015-02-03 | 2017-12-12 | Anaptysbio Inc | ANTIBODIES DIRECTED AGAINST LYMPHOCYTE ACTIVATION GEN 3 (LAG-3) |
MA41867A (en) | 2015-04-01 | 2018-02-06 | Anaptysbio Inc | T-CELL IMMUNOGLOBULIN AND MUCINE PROTEIN 3 ANTIBODIES (TIM-3) |
MY192202A (en) | 2015-10-02 | 2022-08-06 | Hoffmann La Roche | Bispecific antibodies specific for pd1 and tim3 |
US11045547B2 (en) | 2015-12-16 | 2021-06-29 | Merck Sharp & Dohme Corp. | Anti-LAG3 antibodies and antigen-binding fragments |
RU2769282C2 (en) | 2016-06-20 | 2022-03-30 | Кимаб Лимитед | Anti-pd-l1 and il-2 cytokines |
TW202246349A (en) | 2016-10-11 | 2022-12-01 | 美商艾吉納斯公司 | Anti-lag-3 antibodies and methods of use thereof |
KR102583190B1 (en) | 2016-10-13 | 2023-09-26 | 치아타이 티안큉 파마수티컬 그룹 주식회사 | Anti-LAG-3 antibodies and compositions |
WO2018085469A2 (en) | 2016-11-01 | 2018-05-11 | Anaptysbio, Inc. | Antibodies directed against t cell immunoglobulin and mucin protein 3 (tim-3) |
SG11201903835WA (en) | 2016-11-01 | 2019-05-30 | Anaptysbio Inc | Antibodies directed against programmed death- 1 (pd-1) |
AU2018205401A1 (en) | 2017-01-09 | 2019-07-25 | Tesaro, Inc. | Methods of treating cancer with anti-TIM-3 antibodies |
SG11201906192SA (en) | 2017-01-09 | 2019-08-27 | Tesaro Inc | Methods of treating cancer with anti-pd-1 antibodies |
SG11201907208XA (en) | 2017-02-10 | 2019-09-27 | Regeneron Pharma | Radiolabeled anti-lag3 antibodies for immuno-pet imaging |
KR102144317B1 (en) * | 2017-02-22 | 2020-08-18 | 아이-맵 바이오파마 유에스 리미티드 | Anti-LAG-3 antibody and use thereof |
MX2019011770A (en) | 2017-04-03 | 2020-01-09 | Hoffmann La Roche | Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15. |
CN116375876A (en) | 2017-04-05 | 2023-07-04 | 豪夫迈·罗氏有限公司 | Bispecific antibodies that specifically bind PD1 and LAG3 |
MA49042A (en) * | 2017-04-05 | 2020-02-12 | Symphogen As | TARGETING POLYTHERAPIES PD-1, TIM-3 AND LAG-3 |
SI3606954T1 (en) | 2017-04-05 | 2022-10-28 | F. Hoffmann - La Roche Ag | Anti-lag3 antibodies |
CN110799541A (en) * | 2017-04-27 | 2020-02-14 | 特沙诺有限公司 | Antibody agents against lymphocyte activation gene-3 (LAG-3) and uses thereof |
MX2019013755A (en) | 2017-05-18 | 2020-07-20 | Tesaro Inc | Combination therapies for treating cancer. |
CN110720039A (en) | 2017-05-30 | 2020-01-21 | 百时美施贵宝公司 | Treatment of LAG-3 positive tumors |
EP3630842A2 (en) | 2017-05-30 | 2020-04-08 | Bristol-Myers Squibb Company | Compositions comprising a combination of an anti-lag-3 antibody, a pd-1 pathway inhibitor, and an immunotherapeutic agent |
US11723975B2 (en) | 2017-05-30 | 2023-08-15 | Bristol-Myers Squibb Company | Compositions comprising an anti-LAG-3 antibody or an anti-LAG-3 antibody and an anti-PD-1 or anti-PD-L1 antibody |
WO2019046225A1 (en) | 2017-08-30 | 2019-03-07 | Phanes Therapeutics, Inc. | Anti-lag-3 antibodies and uses thereof |
SG11202002113TA (en) | 2017-09-26 | 2020-04-29 | Tesaro Inc | Niraparib formulations |
AU2018338901A1 (en) | 2017-09-30 | 2020-05-07 | Tesaro, Inc. | Combination therapies for treating cancer |
AU2018346688A1 (en) | 2017-10-06 | 2020-04-23 | Tesaro, Inc. | Combination therapies and uses thereof |
WO2019149716A1 (en) | 2018-01-31 | 2019-08-08 | F. Hoffmann-La Roche Ag | Bispecific antibodies comprising an antigen-binding site binding to lag3 |
JP7438180B2 (en) | 2018-03-20 | 2024-02-26 | ウーシー バイオロジクス アイルランド リミテッド | Novel anti-LAG-3 antibody polypeptide |
CN110305215B (en) * | 2018-03-20 | 2023-11-03 | 基石药业 | Novel anti-LAG-3 antibody polypeptides |
CN110343178B (en) * | 2018-04-03 | 2022-07-22 | 上海开拓者生物医药有限公司 | Anti-human LAG-3 monoclonal antibody and application thereof |
CN110615840A (en) * | 2018-06-19 | 2019-12-27 | 信达生物制药(苏州)有限公司 | Fully human anti-LAG-3 antibodies and uses thereof |
WO2020005003A1 (en) * | 2018-06-29 | 2020-01-02 | 주식회사 와이바이오로직스 | Monoclonal antibody specifically binding to lag-3 and use thereof |
SG11202100693UA (en) | 2018-07-26 | 2021-02-25 | Bristol Myers Squibb Co | Lag-3 combination therapy for the treatment of cancer |
TW202024638A (en) | 2018-09-04 | 2020-07-01 | 美商泰沙羅公司 | Methods of treating cancer |
TW202028181A (en) | 2018-10-03 | 2020-08-01 | 美商提薩羅有限公司 | Niraparib salts |
KR20210069678A (en) | 2018-10-03 | 2021-06-11 | 테사로, 인코포레이티드 | Crystalline Form of Niraparib Free Base |
CA3117016A1 (en) | 2018-10-19 | 2020-04-23 | Bristol-Myers Squibb Company | Combination therapy for melanoma |
JP2022534967A (en) | 2019-05-30 | 2022-08-04 | ブリストル-マイヤーズ スクイブ カンパニー | Multiple tumor gene signatures and their uses |
CN114127315A (en) | 2019-05-30 | 2022-03-01 | 百时美施贵宝公司 | Method of identifying subjects suitable for immunooncology (I-O) therapy |
KR20220016157A (en) | 2019-05-30 | 2022-02-08 | 브리스톨-마이어스 스큅 컴퍼니 | Cell localization signatures and combination therapies |
CN115340606B (en) * | 2019-05-31 | 2023-12-19 | 瑞阳(苏州)生物科技有限公司 | Antibody combined with human LAG-3 protein, encoding gene and application thereof |
WO2021024020A1 (en) | 2019-08-06 | 2021-02-11 | Astellas Pharma Inc. | Combination therapy involving antibodies against claudin 18.2 and immune checkpoint inhibitors for treatment of cancer |
KR20220066334A (en) | 2019-09-22 | 2022-05-24 | 브리스톨-마이어스 스큅 컴퍼니 | Quantitative spatial profiling for LAG-3 antagonist therapy |
WO2021092220A1 (en) | 2019-11-06 | 2021-05-14 | Bristol-Myers Squibb Company | Methods of identifying a subject with a tumor suitable for a checkpoint inhibitor therapy |
WO2021092221A1 (en) | 2019-11-06 | 2021-05-14 | Bristol-Myers Squibb Company | Methods of identifying a subject with a tumor suitable for a checkpoint inhibitor therapy |
JP2022553851A (en) | 2019-11-08 | 2022-12-26 | ブリストル-マイヤーズ スクイブ カンパニー | LAG-3 antagonists for the treatment of melanoma |
KR20220129548A (en) | 2019-12-18 | 2022-09-23 | 테사로, 인코포레이티드 | Biopharmaceutical compositions and related methods |
WO2021158938A1 (en) | 2020-02-06 | 2021-08-12 | Bristol-Myers Squibb Company | Il-10 and uses thereof |
EP4178611A1 (en) | 2020-07-07 | 2023-05-17 | BioNTech SE | Therapeutic rna for hpv-positive cancer |
EP4204095A1 (en) | 2020-08-28 | 2023-07-05 | Bristol-Myers Squibb Company | Lag-3 antagonist therapy for hepatocellular carcinoma |
AU2021334361A1 (en) | 2020-08-31 | 2023-05-11 | Bristol-Myers Squibb Company | Cell localization signature and immunotherapy |
EP4232019A1 (en) | 2020-10-23 | 2023-08-30 | Bristol-Myers Squibb Company | Lag-3 antagonist therapy for lung cancer |
WO2022120179A1 (en) | 2020-12-03 | 2022-06-09 | Bristol-Myers Squibb Company | Multi-tumor gene signatures and uses thereof |
WO2022135667A1 (en) | 2020-12-21 | 2022-06-30 | BioNTech SE | Therapeutic rna for treating cancer |
WO2022135666A1 (en) | 2020-12-21 | 2022-06-30 | BioNTech SE | Treatment schedule for cytokine proteins |
TW202245808A (en) | 2020-12-21 | 2022-12-01 | 德商拜恩迪克公司 | Therapeutic rna for treating cancer |
US20220233693A1 (en) | 2020-12-28 | 2022-07-28 | Bristol-Myers Squibb Company | Antibody Compositions and Methods of Use Thereof |
US20220233689A1 (en) | 2020-12-28 | 2022-07-28 | Bristol-Myers Squibb Company | Methods of treating tumors |
CN112852870B (en) * | 2021-01-13 | 2022-03-04 | 北京鼎成肽源生物技术有限公司 | Application of IL-18 gene fusion H2Kk gene in preparation of cell for enhancing cellular immunity, cell and preparation method |
WO2022166987A1 (en) * | 2021-02-08 | 2022-08-11 | 迈威(上海)生物科技股份有限公司 | Antibodies binding lag-3 and use thereof |
CN117858719A (en) | 2021-03-29 | 2024-04-09 | 朱诺治疗学股份有限公司 | Methods of dosing and treatment using a combination of checkpoint inhibitor therapy and CAR T cell therapy |
WO2022240741A1 (en) | 2021-05-12 | 2022-11-17 | Dana-Farber Cancer Institute, Inc. | Lag3 and gal3 inhibitory agents, xbp1, cs1, and cd138 peptides, and methods of use thereof |
AU2022312698A1 (en) | 2021-07-13 | 2024-01-25 | BioNTech SE | Multispecific binding agents against cd40 and cd137 in combination therapy for cancer |
CN113603779B (en) * | 2021-08-18 | 2023-06-02 | 深圳市元谷生物科技有限公司 | Antibody combined with human lymphocyte activation gene 3 (LAG-3) and application thereof |
TW202333802A (en) | 2021-10-11 | 2023-09-01 | 德商拜恩迪克公司 | Therapeutic rna for lung cancer |
IL309227A (en) | 2021-10-29 | 2024-02-01 | Bristol Myers Squibb Co | Lag-3 antagonist therapy for hematological cancer |
WO2023147371A1 (en) | 2022-01-26 | 2023-08-03 | Bristol-Myers Squibb Company | Combination therapy for hepatocellular carcinoma |
WO2023164638A1 (en) | 2022-02-25 | 2023-08-31 | Bristol-Myers Squibb Company | Combination therapy for colorectal carcinoma |
WO2023168404A1 (en) | 2022-03-04 | 2023-09-07 | Bristol-Myers Squibb Company | Methods of treating a tumor |
WO2023170606A1 (en) | 2022-03-08 | 2023-09-14 | Alentis Therapeutics Ag | Use of anti-claudin-1 antibodies to increase t cell availability |
WO2023178329A1 (en) | 2022-03-18 | 2023-09-21 | Bristol-Myers Squibb Company | Methods of isolating polypeptides |
CN114874324B (en) * | 2022-05-13 | 2023-02-03 | 苏州旭光科星抗体生物科技有限公司 | Enzyme-linked immunoassay kit for detecting content of soluble LAG-3 protein and application thereof |
WO2023235847A1 (en) | 2022-06-02 | 2023-12-07 | Bristol-Myers Squibb Company | Antibody compositions and methods of use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20230203156A1 (en) * | 2020-01-29 | 2023-06-29 | Kenjockety Biotechnology, Inc. | Anti-mdr1 antibodies and uses thereof |
Family Cites Families (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4828981A (en) | 1983-08-24 | 1989-05-09 | Synbiotics Corporation | Immunoassays for determining Dirofilaria immitis infection using antiidiotype monoclonal antibody reagents |
GB8601597D0 (en) | 1986-01-23 | 1986-02-26 | Wilson R H | Nucleotide sequences |
CA2096222C (en) | 1990-11-13 | 1998-12-29 | Stephen D. Lupton | Bifunctional selectable fusion genes |
AU6953394A (en) | 1993-05-21 | 1994-12-20 | Targeted Genetics Corporation | Bifunctional selectable fusion genes based on the cytosine deaminase (cd) gene |
US5814618A (en) | 1993-06-14 | 1998-09-29 | Basf Aktiengesellschaft | Methods for regulating gene expression |
US5464758A (en) | 1993-06-14 | 1995-11-07 | Gossen; Manfred | Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters |
MX9605365A (en) | 1994-05-06 | 1997-12-31 | Roussy Inst Gustave | Lag-3 protein soluble polypeptide fractions, method of production, therapeutic composition and anti-idiotype antibody. |
FR2814642B1 (en) | 2000-10-03 | 2005-07-01 | Ass Pour Le Dev De La Rech En | TRANSGENIC MOUSE FOR THE TARGETED RECOMBINATION MEDIATED BY THE MODIFIED CRE-ER |
DE60226641D1 (en) | 2001-12-03 | 2008-06-26 | Amgen Fremont Inc | ANTIBODY CATEGORIZATION BASED ON BINDING CHARACTERISTICS |
EP1897548B1 (en) | 2003-02-28 | 2013-08-14 | The Johns Hopkins University | T cell regulation |
CA2678451A1 (en) | 2007-02-20 | 2008-08-28 | Robert A. Horlick | Somatic hypermutation systems |
EP1987839A1 (en) | 2007-04-30 | 2008-11-05 | I.N.S.E.R.M. Institut National de la Sante et de la Recherche Medicale | Cytotoxic anti-LAG-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease |
EP2044949A1 (en) * | 2007-10-05 | 2009-04-08 | Immutep | Use of recombinant lag-3 or the derivatives thereof for eliciting monocyte immune response |
AR072999A1 (en) | 2008-08-11 | 2010-10-06 | Medarex Inc | HUMAN ANTIBODIES THAT JOIN GEN 3 OF LYMPHOCYTARY ACTIVATION (LAG-3) AND THE USES OF THESE |
CA2863834A1 (en) | 2012-02-06 | 2013-08-15 | Inhibrx Llc | Cd47 antibodies and methods of use thereof |
UY34887A (en) | 2012-07-02 | 2013-12-31 | Bristol Myers Squibb Company Una Corporacion Del Estado De Delaware | OPTIMIZATION OF ANTIBODIES THAT FIX THE LYMPHOCYTE ACTIVATION GEN 3 (LAG-3) AND ITS USES |
US9574013B2 (en) | 2012-12-07 | 2017-02-21 | Vanderbilt University | Antibodies against factor XII and uses thereof |
EP2961388B1 (en) | 2013-03-01 | 2019-04-24 | Astex Pharmaceuticals, Inc. | Drug combinations |
KR101763352B1 (en) * | 2013-03-15 | 2017-07-31 | 글락소스미스클라인 인털렉츄얼 프로퍼티 디벨로프먼트 리미티드 | Anti-lag-3 binding proteins |
EP3119913B1 (en) | 2014-03-21 | 2021-01-06 | The Brigham and Women's Hospital, Inc. | Methods and compositions for treatment of immune-related diseases or disorders and/or therapy monitoring |
US20150307620A1 (en) | 2014-04-16 | 2015-10-29 | University Of Connecticut | Linked immunotherapeutic agonists that costimulate multiple pathways |
US20170184604A1 (en) | 2014-05-22 | 2017-06-29 | The General Hospital Corporation | Dd1alpha receptor and uses thereof in immune disorders |
ES2808153T3 (en) | 2014-10-31 | 2021-02-25 | Mereo Biopharma 5 Inc | Combination therapy for disease treatment |
MA41463A (en) | 2015-02-03 | 2017-12-12 | Anaptysbio Inc | ANTIBODIES DIRECTED AGAINST LYMPHOCYTE ACTIVATION GEN 3 (LAG-3) |
TWI773646B (en) | 2015-06-08 | 2022-08-11 | 美商宏觀基因股份有限公司 | Lag-3-binding molecules and methods of use thereof |
-
2016
- 2016-02-02 MA MA041463A patent/MA41463A/en unknown
- 2016-02-03 EA EA201791742A patent/EA201791742A1/en unknown
- 2016-02-03 SG SG11201706199RA patent/SG11201706199RA/en unknown
- 2016-02-03 WO PCT/US2016/016424 patent/WO2016126858A2/en active Application Filing
- 2016-02-03 KR KR1020177024728A patent/KR20170116067A/en not_active Application Discontinuation
- 2016-02-03 MX MX2017009904A patent/MX2017009904A/en unknown
- 2016-02-03 EP EP16747220.8A patent/EP3253798A4/en not_active Ceased
- 2016-02-03 EP EP21198994.2A patent/EP4011914A1/en active Pending
- 2016-02-03 US US15/548,405 patent/US10836824B2/en active Active
- 2016-02-03 CA CA2975753A patent/CA2975753A1/en active Pending
- 2016-02-03 BR BR112017016638-0A patent/BR112017016638B1/en active IP Right Grant
- 2016-02-03 CN CN202210381152.5A patent/CN114634573A/en active Pending
- 2016-02-03 SG SG10201907161YA patent/SG10201907161YA/en unknown
- 2016-02-03 JP JP2017540834A patent/JP6949713B2/en active Active
- 2016-02-03 CN CN201680020117.3A patent/CN107428836A/en active Pending
-
2017
- 2017-07-30 IL IL253718A patent/IL253718B/en unknown
- 2017-07-31 MX MX2022000112A patent/MX2022000112A/en unknown
- 2017-08-04 ZA ZA2017/05303A patent/ZA201705303B/en unknown
-
2018
- 2018-05-17 HK HK18106454.6A patent/HK1246814A1/en unknown
- 2018-05-25 HK HK18106890.8A patent/HK1247618A1/en unknown
-
2019
- 2019-05-01 US US16/400,198 patent/US20190256596A1/en not_active Abandoned
-
2020
- 2020-10-19 US US17/074,269 patent/US20210095026A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20230203156A1 (en) * | 2020-01-29 | 2023-06-29 | Kenjockety Biotechnology, Inc. | Anti-mdr1 antibodies and uses thereof |
Non-Patent Citations (5)
Title |
---|
Alahmari et al., (Cancer Research, 2023, v.83, v.8 LB194 * |
Cebada et al ( Expert Opinion on Therapeutic Patents, 2020, v.30, pages 487-494 * |
Mestas et al ( J. of Immunology, 2004, 172, pages 2731-238 * |
Shanks et al ( Philosophy, Ethics and Humanities in Medicine, 2009, v.4, pages 1-20 * |
Teuveson et al., ( Immun. Review 1993, N136, pages 101-107 * |
Also Published As
Publication number | Publication date |
---|---|
SG10201907161YA (en) | 2019-09-27 |
EP4011914A1 (en) | 2022-06-15 |
KR20170116067A (en) | 2017-10-18 |
IL253718B (en) | 2021-08-31 |
US10836824B2 (en) | 2020-11-17 |
CA2975753A1 (en) | 2016-08-11 |
IL253718A0 (en) | 2017-09-28 |
BR112017016638A2 (en) | 2018-06-19 |
JP6949713B2 (en) | 2021-10-13 |
HK1247618A1 (en) | 2018-09-28 |
ZA201705303B (en) | 2022-12-21 |
US20190256596A1 (en) | 2019-08-22 |
EP3253798A2 (en) | 2017-12-13 |
BR112017016638B1 (en) | 2023-11-07 |
MA41463A (en) | 2017-12-12 |
MX2022000112A (en) | 2022-02-10 |
MX2017009904A (en) | 2018-06-27 |
WO2016126858A3 (en) | 2016-09-29 |
EA201791742A1 (en) | 2018-01-31 |
HK1246814A1 (en) | 2018-09-14 |
WO2016126858A2 (en) | 2016-08-11 |
CN107428836A (en) | 2017-12-01 |
EP3253798A4 (en) | 2018-07-25 |
SG11201706199RA (en) | 2017-08-30 |
US20180127496A1 (en) | 2018-05-10 |
JP2018505674A (en) | 2018-03-01 |
CN114634573A (en) | 2022-06-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210095026A1 (en) | Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3) | |
US20230331843A1 (en) | Antibodies Directed Against Programmed Death-1(PD-1) | |
US11352427B2 (en) | Antibodies directed against T Cell immunoglobulin and mucin protein 3 (TIM-3) | |
EA040513B1 (en) | ANTIBODIES DIRECTED AGAINST LYMPHOCYTE ACTIVATION GENE-3 (LAG-3) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ANAPTYSBIO, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JUN, HELEN TONI;KEHRY, MARILYN;BOWERS, PETER;AND OTHERS;REEL/FRAME:054102/0422 Effective date: 20160205 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |