CN118871115A

CN118871115A - IL-18BP antagonist antibodies and their use as monotherapy and combination therapy in cancer treatment

Info

Publication number: CN118871115A
Application number: CN202380025365.7A
Authority: CN
Inventors: N·P·尼尔森; A·M·奇亚森; A·梅纳切姆; E·奥菲尔; O·莱德曼; T·弗里德曼-克弗; M·加尔佩林; H·G·蒂勒曼; D·布拉特; G·科乔卡鲁; A·托珀里克; A·诺维克; Z·埃尔利希; Z·阿尔特伯; E·塔蒂罗夫斯基; M·珀皮涅尔; I·塞沃; N·科恩
Original assignee: Compugen Ltd
Current assignee: Compugen Ltd
Priority date: 2022-03-15
Filing date: 2023-03-15
Publication date: 2024-10-29

Abstract

本发明涉及抗IL18‑BP抗体或其用途。本发明涉及利用例如本文所描述的免疫检查点抑制剂抗体的单一疗法和组合疗法。The present invention relates to anti-IL18-BP antibodies or uses thereof. The present invention relates to monotherapy and combination therapy utilizing immune checkpoint inhibitor antibodies such as those described herein.

Description

IL-18BP antagonist antibodies and their use in monotherapy and in combination therapy in the treatment of cancer

The present application claims priority from U.S. patent application Ser. No. 63/320,202, filed on 3/15, 2022, U.S. patent application Ser. No. 63/351,242, filed on 10, 6, 2022, and U.S. patent application Ser. No. 63/478,898, filed on 6, 1, 2023, which are hereby incorporated by reference in their entirety.

Background

Interleukin 18 (IL-18) is a pro-inflammatory cytokine that stimulates T cells, NK cells, and bone marrow cells. IL-18 has been proposed as an immunotherapeutic agent for the treatment of cancer in view of its ability to stimulate anti-tumor immune cells. However, IL-18 has limited clinical efficacy and thus there is a need for compositions and methods that provide effective IL-18 signaling activity to treat and prevent cancer and other diseases and disorders.

Interleukin 18 binding protein (IL 18-BP) binds IL18, preventing IL18 from binding to its receptor, and thus acts as an inhibitor of the pro-inflammatory cytokine IL 18. IL18-BP inhibits IL 18-induced T-cell and NK-cell activation and proliferation, as well as the production of pro-inflammatory cytokines, thereby reducing T-cell and NK-cell activity and reducing T-helper type 1 immune responses.

It is an object of the present invention to provide anti-IL 18-BP antibodies or use in the treatment of diseases. The present invention meets this need by providing anti-IL 18-BP antibodies (including antigen binding fragments), particularly anti-IL 18-BP antibodies that block IL18-BP, which are useful in the treatment of diseases such as cancer.

Disclosure of Invention

The present invention provides compositions and methods related to anti-IL 18-BP antibodies.

In some embodiments, the invention provides a composition comprising an anti-IL 18-BP (interleukin-18 binding protein) antibody for activating T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells and/or congenital lymphoid cells (ILCs) and/or modulating bone marrow cells for use in treating cancer, wherein the antibody antagonizes at least one immunosuppressive effect of IL18-BP, optionally wherein the anti-IL 18-BP antibody blocks IL18: IL18-BP binding interaction, optionally wherein the anti-IL 18-BP antibody exhibits a binding affinity of less than 1 pM.

In some embodiments, the composition comprises an anti-IL 18-BP antibody, wherein the anti-IL 18-BP competes for binding with an antibody that binds to the secretory chain of human IL18-BP of SEQ ID NO. 254 and/or human IL18-BP of SEQ ID NO. 255 and/or competes for binding with IL 18.

In some embodiments, the composition comprises an anti-IL 18-BP antibody, wherein the anti-IL 18-BP competes for binding with an antibody as described in US 8436148, WO 2019213686, WO 200107480, WO 2019051015, WO 2014126277A1, WO 2012177595, US20140364341, and/or WO 2018060447.

In some embodiments, the composition comprises an anti-IL 18-BP antibody, wherein the antibody comprises: vhCDR1, vhCDR2, vhCDR, vlCDR1, vlCDR2, and vlCDR3 sequences selected from the group consisting of:

i. vhCDR1(SEQ ID NO:1)、vhCDR2(SEQ ID NO:32)、vhCDR3(SEQ ID NO:3)、vlCDR1(SEQ ID NO:4)、vlCDR2(SEQ ID NO:5) and vlCDR3 (SEQ ID NO: 6) sequence (66650) of FIG. 1A;

vhCDR1(SEQ ID NO:7)、vhCDR2(SEQ ID NO:8)、vhCDR3(SEQ ID NO:9)、vlCDR1(SEQ ID NO:10)、vlCDR2(SEQ ID NO:11) and vlCDR (SEQ ID NO: 12) sequence (66670) of FIG. 1B;

vhCDR1(SEQ ID NO:13)、vhCDR2(SEQ ID NO:14)、vhCDR3(SEQ ID NO:15)、vlCDR1(SEQ ID NO:16)、vlCDR2(SEQ ID NO:17) and vlCDR (SEQ ID NO: 18) sequence (66692) of FIG. 1C;

vhCDR1(SEQ ID NO:19)、vhCDR2(SEQ ID NO:20)、vhCDR3(SEQ ID NO:21)、vlCDR1(SEQ ID NO:22)、vlCDR2(SEQ ID NO:23) and vlCDR (SEQ ID NO: 24) sequence (66716) of FIG. 1D;

v. the vhCDR1(SEQ ID NO:25)、vhCDR2(SEQ ID NO:26)、vhCDR3(SEQ ID NO:27)、vlCDR1(SEQ ID NO:28)、vlCDR2(SEQ ID NO:29) and vlCDR (SEQ ID NO: 30) sequences of FIG. 1E (66650);

vhCDR1(SEQ ID NO:31)、vhCDR2(SEQ ID NO:32)、vhCDR3(SEQ ID NO:33)、vlCDR1(SEQ ID NO:34)、vlCDR2(SEQ ID NO:35) and vlCDR (SEQ ID NO: 36) sequence (66670) of FIG. 1F;

vhCDR1(SEQ ID NO:37)、vhCDR2(SEQ ID NO:38)、vhCDR3(SEQ ID NO:39)、vlCDR1(SEQ ID NO:40)、vlCDR2(SEQ ID NO:41) and vlCDR (SEQ ID NO: 42) sequences (66692) of FIG. 1G;

vhCDR1(SEQ ID NO:43)、vhCDR2(SEQ ID NO:44)、vhCDR3(SEQ ID NO:45)、vlCDR1(SEQ ID NO:46)、vlCDR2(SEQ ID NO:47) and vlCDR (SEQ ID NO: 48) sequences (66716) of FIG. 1H;

x. vhCDR1(SEQ ID NO:844)、vhCDR2(SEQ ID NO:845)、vhCDR3(SEQ ID NO:846)、vlCDR1(SEQ ID NO:847)、vlCDR2(SEQ ID NO:848) and vlCDR (SEQ ID NO: 849) sequences (66650) of FIG. 1I;

vhCDR1(SEQ ID NO:850)、vhCDR2(SEQ ID NO:851)、vhCDR3(SEQ ID NO:852)、vlCDR1(SEQ ID NO:853)、vlCDR2(SEQ ID NO:854) and vlCDR (SEQ ID NO: 855) sequences (66670) of FIG. 1J;

The vhCDR1(SEQ ID NO:856)、vhCDR2(SEQ ID NO:857)、vhCDR3(SEQ ID NO:858)、vlCDR1(SEQ ID NO:859)、vlCDR2(SEQ ID NO:860) and vlCDR (SEQ ID NO: 861) sequences of FIG. 1K (66692);

vhCDR1(SEQ ID NO:862)、vhCDR2(SEQ ID NO:863)、vhCDR3(SEQ ID NO:864)、vlCDR1(SEQ ID NO:865)、vlCDR2(SEQ ID NO:866) and vlCDR (SEQ ID NO: 867) sequences (66716) of FIG. 1L;

The vhCDR1(SEQ ID NO:55)、vhCDR2(SEQ ID NO:56)、vhCDR3(SEQ ID NO:57)、vlCDR1(SEQ ID NO:60)、vlCDR2(SEQ ID NO:61) and vlCDR (SEQ ID NO: 62) sequences of FIG. 2A (71709);

xv. the vhCDR1(SEQ ID NO:65)、vhCDR2(SEQ ID NO:66)、vhCDR3(SEQ ID NO:67)、vlCDR1(SEQ ID NO:70)、vlCDR2(SEQ ID NO:71) and vlCDR3 (SEQ ID NO: 72) sequences of FIG. 2B (71719);

xvi. vhCDR1(SEQ ID NO:75)、vhCDR2(SEQ ID NO:76)、vhCDR3(SEQ ID NO:77)、vlCDR1(SEQ ID NO:80)、vlCDR2(SEQ ID NO:81) and vlCDR (SEQ ID NO: 82) sequences (71720) of FIG. 2C;

xvii. vhCDR1(SEQ ID NO:85)、vhCDR2(SEQ ID NO:86)、vhCDR3(SEQ ID NO:87)、vlCDR1(SEQ ID NO:90)、vlCDR2(SEQ ID NO:91) and vlCDR (SEQ ID NO: 92) sequences (71722) of FIG. 2D;

xviii. vhCDR1(SEQ ID NO:95)、vhCDR2(SEQ ID NO:96)、vhCDR3(SEQ ID NO:97)、vlCDR1(SEQ ID NO:100)、vlCDR2(SEQ ID NO:101) and vlCDR (SEQ ID NO: 102) sequence (71701) of FIG. 2E;

vhCDR1(SEQ ID NO:105)、vhCDR2(SEQ ID NO:106)、vhCDR3(SEQ ID NO:107)、vlCDR1(SEQ ID NO:110)、vlCDR2(SEQ ID NO:111) and vlCDR (SEQ ID NO: 112) sequence (71663) of FIG. 2F;

xx. the vhCDR1(SEQ ID NO:115)、vhCDR2(SEQ ID NO:116)、vhCDR3(SEQ ID NO:117)、vlCDR1(SEQ ID NO:120)、vlCDR2(SEQ ID NO:121) and vlCDR (SEQ ID NO: 122) sequences of FIG. 2G (71662);

xxi. vhCDR1(SEQ ID NO:125)、vhCDR2(SEQ ID NO:126)、vhCDR3(SEQ ID NO:127)、vlCDR1(SEQ ID NO:130)、vlCDR2(SEQ ID NO:131) and vlCDR (SEQ ID NO: 132) sequences (66692) of FIG. 2H;

xxii. vhCDR1(SEQ ID NO:135)、vhCDR2(SEQ ID NO:136)、vhCDR3(SEQ ID NO:137)、vlCDR1(SEQ ID NO:140)、vlCDR2(SEQ ID NO:141) and vlCDR (SEQ ID NO: 142) sequences of FIG. 2I (71710);

xxiii the vhCDR1(SEQ ID NO:145)、vhCDR2(SEQ ID NO:146)、vhCDR3(SEQ ID NO:147)、vlCDR1(SEQ ID NO:150)、vlCDR2(SEQ ID NO:151) and vlCDR (SEQ ID NO: 152) sequences (71717) of FIG. 2J;

xxiv the vhCDR1(SEQ ID NO:155)、vhCDR2(SEQ ID NO:156)、vhCDR3(SEQ ID NO:157)、vlCDR1(SEQ ID NO:160)、vlCDR2(SEQ ID NO:161) and vlCDR (SEQ ID NO: 162) sequences (71739) of FIG. 2K;

xxv. vhCDR1(SEQ ID NO:165)、vhCDR2(SEQ ID NO:166)、vhCDR3(SEQ ID NO:167)、vlCDR1(SEQ ID NO:170)、vlCDR2(SEQ ID NO:171) and vlCDR (SEQ ID NO: 172) sequences (71736) of FIG. 2L;

xxvi. vhCDR1(SEQ ID NO:175)、vhCDR2(SEQ ID NO:176)、vhCDR3(SEQ ID NO:177)、vlCDR1(SEQ ID NO:180)、vlCDR2(SEQ ID NO:181) and vlCDR (SEQ ID NO: 182) sequences of FIG. 2M (71707);

xxvii. vhCDR1(SEQ ID NO:185)、vhCDR2(SEQ ID NO:186)、vhCDR3(SEQ ID NO:187)、vlCDR1(SEQ ID NO:190)、vlCDR2(SEQ ID NO:191) and vlCDR (SEQ ID NO: 192) sequences of FIG. 2N (66716);

xxviii. vhCDR1(SEQ ID NO:195)、vhCDR2(SEQ ID NO:196)、vhCDR3(SEQ ID NO:197)、vlCDR1(SEQ ID NO:200)、vlCDR2(SEQ ID NO:201) and vlCDR (SEQ ID NO: 202) sequences of FIG. 2O (71728);

xxix. vhCDR1(SEQ ID NO:205)、vhCDR2(SEQ ID NO:206)、vhCDR3(SEQ ID NO:207)、vlCDR1(SEQ ID NO:210)、vlCDR2(SEQ ID NO:211) and vlCDR (SEQ ID NO: 212) sequences (71741) of FIG. 2P;

xxx. vhCDR1(SEQ ID NO:215)、vhCDR2(SEQ ID NO:216)、vhCDR3(SEQ ID NO:217)、vlCDR1(SEQ ID NO:220)、vlCDR2(SEQ ID NO:221) and vlCDR (SEQ ID NO: 222) sequences of FIG. 2Q (71742);

xxxi. vhCDR1(SEQ ID NO:225)、vhCDR2(SEQ ID NO:226)、vhCDR3(SEQ ID NO:227)、vlCDR1(SEQ ID NO:230)、vlCDR2(SEQ ID NO:231) and vlCDR (SEQ ID NO: 232) sequences (71744) of FIG. 2R;

xxxii. vhCDR1(SEQ ID NO:235)、vhCDR2(SEQ ID NO:236)、vhCDR3(SEQ ID NO:237)、vlCDR1(SEQ ID NO:240)、vlCDR2(SEQ ID NO:241) and vlCDR (SEQ ID NO: 242) sequences (71753) of FIG. 2S; and

Xxxiii. vhCDR1(SEQ ID NO:245)、vhCDR2(SEQ ID NO:246)、vhCDR3(SEQ ID NO:247)、vlCDR1(SEQ ID NO:250)、vlCDR2(SEQ ID NO:251) and vlCDR (SEQ ID NO: 252) sequences (71755) of FIG. 2T.

In some embodiments, the composition comprises and antibody, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of an antibody selected from the group consisting of:

i. the heavy chain variable domain (SEQ ID NO: 54) and the light chain variable domain (SEQ ID NO: 59) of FIG. 2A (71709);

heavy chain variable domain (SEQ ID NO: 64) and light chain variable domain (SEQ ID NO: 69) (71719) of FIG. 2B;

Heavy chain variable domain (SEQ ID NO: 74) and light chain variable domain (SEQ ID NO: 79) of FIG. 2C (71720);

Heavy chain variable domain (SEQ ID NO: 84) and light chain variable domain (SEQ ID NO: 89) of FIG. 2D (71722);

v. the heavy chain variable domain (SEQ ID NO: 94) and the light chain variable domain (SEQ ID NO: 99) of FIG. 2E (71701);

vi heavy chain variable domain (SEQ ID NO: 104) and light chain variable domain (SEQ ID NO: 109) (71663) of FIG. 2F;

heavy chain variable domain (SEQ ID NO: 114) and light chain variable domain (SEQ ID NO: 119) of FIG. 2G (71662);

heavy chain variable domain (SEQ ID NO: 124) and light chain variable domain (SEQ ID NO: 129) (66692) of FIG. 2H;

ix. heavy chain variable domain (SEQ ID NO: 134) and light chain variable domain (SEQ ID NO: 139) of FIG. 2I (71710);

x. heavy chain variable domain (SEQ ID NO: 144) and light chain variable domain (SEQ ID NO: 149) of FIG. 2J (71717);

heavy chain variable domain (SEQ ID NO: 154) and light chain variable domain (SEQ ID NO: 159) (71739) of FIG. 2K;

The heavy chain variable domain (SEQ ID NO: 164) and the light chain variable domain (SEQ ID NO: 169) of FIG. 2L (71736);

the heavy chain variable domain (SEQ ID NO: 174) and the light chain variable domain (SEQ ID NO: 179) of FIG. 2M (71707);

Heavy chain variable domain (SEQ ID NO: 184) and light chain variable domain (SEQ ID NO: 189) of FIG. 2N (66716);

xv. the heavy chain variable domain (SEQ ID NO: 194) and the light chain variable domain (SEQ ID NO: 199) of FIG. 2O (71728);

xvi. the heavy chain variable domain (SEQ ID NO: 204) and the light chain variable domain (SEQ ID NO: 209) of FIG. 2P (71741);

xvii A heavy chain variable domain (SEQ ID NO: 214) and a light chain variable domain (SEQ ID NO: 219) of FIG. 2Q (71742);

xviii the heavy chain variable domain (SEQ ID NO: 224) and the light chain variable domain (SEQ ID NO: 229) (71744) of FIG. 2R;

xix. heavy chain variable domain (SEQ ID NO: 234) and light chain variable domain (SEQ ID NO: 239) (71753) of FIG. 2S; and

Xx. heavy chain variable domain (SEQ ID NO: 244) and light chain variable domain (SEQ ID NO: 249) of FIG. 2T (71755).

In some embodiments, the antibody comprises a CH 1-hinge-CH 2-CH3 region from human IgG1, igG2, igG3, or IgG4, wherein the hinge region optionally comprises a mutation.

In some embodiments, the antibody comprises a CH 1-hinge-CH 2-CH3 region from human IgG 4.

In some embodiments, the hinge region comprises a mutation.

In some embodiments, the antibody comprises the CL region of a human kappa 2 light chain.

In some embodiments, the antibody comprises the CL region of the human λ2 light chain.

In some embodiments, the antibody comprises:

i. a heavy chain variable domain that is capable of binding to a heavy chain, the heavy chain variable domain comprises:

a) CDR-H1 having the sequence Y-T-F-X-X2-Y-A-X3-H, wherein X is N, R, D, G or K; x2 is S, H, I or Q; x3 is M or V;

b) CDR-H2 having the sequence W-I-H-A-G-T-G-X-T-X2-Y-S-Q-K-F-Q-G, wherein X is N, A or V; x2 is K or LW-I-H; and

C) CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is S or E; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S-L-E-S; and

C) CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is S, V, Y, L or Q; x2 is F, S or G.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence G-T-F-X-X2-Y-X3-I-S, wherein X is S or N; x2 is E or S; x3 is V or P

B) Ext> CDRext> -ext> Hext> 2ext> havingext> theext> sequenceext> Gext> -ext> Iext> -ext> Iext> -ext> Pext> -ext> Gext> -ext> Aext> -ext> Gext> -ext> Text> -ext> Aext> -ext> Xext> -ext> Yext> -ext> Aext> -ext> Qext> -ext> Kext> -ext> Fext> -ext> Qext> -ext> Gext>,ext> whereinext> Xext> isext> Next> orext> IGext> -ext> Iext> -ext> Pext> -ext> Xext> -ext> Xext> 2ext> -ext> Gext> -ext> Xext> 3ext> -ext> Aext> -ext> Xext> 4ext> -ext> Yext> -ext> Aext> -ext> Qext> -ext> Kext> -ext> Fext> -ext> Qext> -ext> Gext>,ext> whereinext> Xext> isext> Gext> orext> Iext>;ext> X2 is S or A; x3 is T or S, X4 is N or I; and

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S or F; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-V-Y-X-X2-P-W-T, wherein X is S or R; x2 is L or FQ-.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence F-T-F-X-N-X2-A-M-SF-T-F-X-N-X2-X3-M-S, wherein X is G or D or S; x2 is T or V or Y;

b) CDR-H2 having the sequence A-I-S-X-X1-X2-G-S-T-Y-Y-A-D-S-V-K-GA-I-S-A-N-A-G-S-T-Y-Y-A-D-S-V-K-G, wherein X is G or A; x2 is N or S; x3 is A or G; and

C) CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y; a light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-H-A-X-X1-F-P-Y-TQ-H-A-L-X-F-P-Y-T, wherein X is Y or L; x1 is S or F.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence G-S-I-S-S-X-X2-Y-X3-W-G, wherein X is S or P; x2 is E or D; x3 is G, Y or P;

b) CDR-H2 having the sequence S-I-X-X2-X3-G-X4-T-Y-Y-N-P-S-L-K-S, wherein X is Y or V; x2 is Y or N; x3 is Q or S; x4 is S or A; and

C) CDR-H3 having the sequence A-R-G-P-X-R-Q-X2-F-D-Y, wherein X is Y or H and X2 is V or L; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is S or F; x2 is S or V.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence Y-T-F-X-X2-Y-A-X3-H, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

b) CDR-H2 having the sequence W-I-H-A-G-T-G-X-T-X2-Y-S-Q-K-F-Q-G, wherein X is any amino acid; x2 is any amino acid; and

C) CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is any amino acid; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S-L-E-S; and

C) CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence G-T-F-X-X2-Y-X3-I-S, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

b) CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is G or Y and X2 is A or S; x3 is N, I or V; and

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is any amino acid; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-V-Y-X-X2-P-W-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence F-T-F-X-N-X2-A-M-S, wherein X is any amino acid; x2 is any amino acid;

b) CDR-H2 having the sequence A-I-S-X-X1-X2-G-S-T-Y-Y-A-D-S-V-K-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid; and

C) CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is any amino acid

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-H-A-X-X1-F-P-Y-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence G-S-I-S-S-X-X2-Y-X3-W-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

b) CDR-H2 having the sequence S-I-X-X2-X3-G-X4-T-Y-Y-N-P-S-L-K-S, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid; x4 is any amino acid; and

C) CDR-H3 having the sequence A-R-G-P-X-R-Q-X2-F-D-Y, wherein X is any amino acid and X2 is any amino acid; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence Y-T-F-X-X2-Y-A-X3-H, wherein X is N, R, D, G, T, Q, S, A or K; x2 is S, H, I, N, L, Y or Q; x3 is M or V;

b) CDR-H2 having the sequence X-I-X2-A-G-X3-X4-X5-T-X6-Y-S-Q-K-F-Q-G, wherein X is W or Y; x2 is H or N; x3 is S, T or A; x4 is G or A; x5 is N, A, T or V; x6 is E, K or L; and

C) CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is S, L, A, K or E; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S-E-S, wherein X is L or S; and

C) CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is S, V, Y, L, T or Q; x2 is F, S, Y or G.

In some embodiments, the antibody comprises:

B) CDR-H2 having the sequence G-I-I-P-X-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is G, S, I or Y; x2 is A, V or S; x3 is N, I or V; and

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-X-Y-X2-X3-P-W-T, wherein X is V or L; x2 is S or R; x3 is L, I or F.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence F-T-F-X-X2-X3-X4-M-S, wherein X is G, S, P or D or S; x2 is N, S or P; x3 is T, V or Y; x4 is A, H or I;

b) CDR-H2 having the sequence A-I-S-X-X2-X3-X4-X5-T-X6-Y-A-D-S-V-K-G, wherein X is G or A; x2 is N, T, E or S; x3 is A or G; x4 is A or G; x5 is S or G; x6 is Y or F; and

a) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-H-X-X2-X3-F-P-Y-T, wherein X is A or G; x2 is Y, R or L; x3 is S, R, L or F.

In some embodiments, the antibody comprises:

a) CDR-H1 having the sequence G-S-I-X-S-X2-X3-Y-X4-W-X5, wherein X is S or F; x2 is S or P; x3 is E or D; x4 is G, P or Y; x5 is G or S;

b) CDR-H2 having the sequence X-I-X2-X3-X4-G-X5-T-Y-Y-N-P-S-L-K-S, wherein X is S or V; x2 is Y, V, F or A; x3 is Y, F or N; x4 is Q, A or S; x5 is S, A or N; and

C) CDR-H3 having the sequence A-R-G-P-X-R-Q-X2-F-D-Y, wherein X is Y, H or F; x2 is V or L; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is S, N, W or F; x2 is S or V.

In some embodiments, the antibody comprises:

i) vhCDR1, vhCDR2 and vhCDR3 from VH1-03.66650, VH1-69.66670, VH3-23.66692 or VH 1-39.66716; and

Ii) vlCDR, vlCDR2 and vlCDR3 from VH1-03.66650, VH1-69.66670, VH3-23.66692 or VH1-39.66716, vlCDR, vlCDR2 and vlCDR3 from VH1-03.66650, VH1-69.66670, VH3-23.66692 or VH 1-39.66716.

In some embodiments, the antibody comprises:

i) vhCDR1, vhCDR2 and vhCDR3 from VH1-03.66650、VH1-69.66670、VH3-23.66692、VH1-39.66716、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755; and

Ii) vlCDR, vlCDR2 and vlCDR3 from VL-κ-1-5、VL-κ-1-12、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755; wherein optionally, the CDR comprises 0 to 4 substituents, and wherein no individual CDR comprises more than 1 substituent, and wherein the vhCDR and vlCDR3 do not comprise substituents.

In some embodiments, the anti-IL 18-BP antibody comprises:

i) Comprising a heavy chain variable domain exhibiting a sequence at least 90%, at least 95% or at least 98% identity to the heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vhCDR comprises no more than 1 substituent, and wherein the vhCDR3 does not comprise a substituent, and

Ii) comprises a light chain variable domain that exhibits a sequence at least 90%, at least 95% or at least 98% identity to the light chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vlCDR comprises no more than 1 substituent, and wherein the vlCDR3 does not comprise a substituent.

In some embodiments, the anti-IL 18-BP antibody comprises:

i) Comprising the heavy chain variable domains of vhCDR, vhCDR2 and vhCDR from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and wherein said heavy chain variable domain comprises a sequence exhibiting at least 90% identity to the heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vhCDR comprises no more than 1 substituent, and wherein the vhCDR3 does not comprise a substituent, and

Ii) comprises the light chain variable domains of vlCDR, vlCDR and vlCDR3 from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and wherein said light chain variable domain comprises a sequence exhibiting at least 90% identity to the light chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vlCDR comprises no more than 1 substituent, and wherein the vlCDR3 does not comprise a substituent.

In some embodiments, the antibody comprises the heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and the light chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755.

In some embodiments, the hinge region comprises a mutation.

In some embodiments, the antibody comprises:

a) Heavy chain variable domain ：VH1-03.66650、VH1-69.66670、VH3-23.66692、VH1-39.66716、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755 comprising antibodies vhCDR1, vhCDR2, and vhCDR3 from the group consisting of

B) Light chain variable domains ：VL-κ-1-5、VL-κ-1-12、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755 comprising vlCDR, vlCDR2, and vlCDR3 from antibodies selected from the group consisting of.

In some embodiments, the antibody comprises:

B) A light chain variable domain ：VL-κ-1-5、VL-κ-1-12、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755 comprising antibodies vlCDR1, vlCDR2, and vlCDR3 from the group consisting of; and

Optionally, 1) wherein each CDR individually comprises 0 to 4 substituents, and wherein no individual CDR comprises more than 1 substituent, and wherein the vhCDR and vlCDR do not comprise substituents, 2) each CDR individually comprises 1 substituent, or 3) wherein each individual vhCDR comprises no more than 1 substituent, and wherein the vhCDR3 does not comprise a substituent.

In some embodiments, the hinge region comprises a mutation.

In some embodiments, the antibody competes for binding with the antibody of any one of the preceding claims.

The invention also provides a method of treating cancer in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the cancer is treated.

The invention also provides a method of treating cancer in a patient, the method comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody activates T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells and/or congenital lymphoid cells (ILCs) and/or modulates bone marrow cells, and wherein the cancer is treated.

The invention also provides a method of activating T cells of a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the T cells are activated.

The invention also provides a method of activating NK cells of a patient, comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein said NK cells are activated.

The invention also provides a method of activating NKT cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the NKT cells are activated.

The invention also provides a method of modulating bone marrow cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the bone marrow cells are modulated.

The invention also provides a method of activating dendritic cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the dendritic cells are activated.

The invention also provides a method of activating dendritic cells in a patient comprising administering an anti-IL 18-BP antibody according to any of the preceding claims, and wherein the MAIT T cells are activated,

The invention also provides a method of activating dendritic cells in a patient comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the γδ T cells are activated.

The invention also provides a method of activating ILC cells of a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the ILC cells are activated.

The invention also provides a method of increasing IL-18-mediated immunostimulatory activity in a Tumor Microenvironment (TME) and/or a lymph node comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody increases IL-18-mediated immunostimulatory activity in the TME and/or lymph node.

The invention also provides a method of restoring IL-18 activity on a T cell, NK cell, NKT cell, bone marrow cell, dendritic cell, MAIT T cell, γδ T cell and/or congenital lymphoid cell (ILC), the method comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody restores activity on a T cell, NK cell, NKT cell, bone marrow cell, dendritic cell, MAIT T cell, γδ T cell and/or congenital lymphoid cell (ILC).

In some embodiments, the anti-IL 18-BP antibody is administered as a stable liquid pharmaceutical formulation.

In some embodiments, the T cell is a cytotoxic T Cell (CTL).

The invention also provides a method according to claim 47, wherein said T cells are selected from the group consisting of CD4 ⁺ T cells and CD8 ⁺ T cells.

In some embodiments, the tumor growth inhibition of the subject for treatment is increased by at least about 10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、125％、150％、175％、200％、225％、250％、275％、300％、325％、350％、375％、400％、425％、450％、475％、500％、525％、550％、575％、600％、625％、650％、675％、700％、725％、750％、775％、800％、825％、850％、875％、900％、925％、950％、975％ or 1000% as compared to a control group or untreated patient.

The method of treatment according to any one of claims 40-54, wherein the subject for treatment exhibits a reduction in tumor growth of at least about 10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、125％、150％、175％、200％、225％、250％、275％、300％、325％、350％、375％、400％、425％、450％、475％、500％、525％、550％、575％、600％、625％、650％、675％、700％、725％、750％、775％、800％、825％、850％、875％、900％、925％、950％、975％ or 1000% compared to a control group or untreated patient.

The invention also provides a method as described herein, wherein the NK cells are cd16+ lymphocytes.

The invention also provides a method as described herein, wherein the NK cells are cd56+ NK cells.

The invention also provides a method as described herein, wherein the activation is measured as an increase in expression of one or more activation markers.

The invention also provides a method as described herein, wherein the activation marker is selected from the group consisting of CD107a, CD137, CD69, granzyme and perforin.

The invention also provides a method as described herein, wherein the activation is measured as an increase in proliferation of said NK cells.

The invention also provides a method as described herein, wherein the activation is measured as an increase in secretion of one or more cytokines.

The invention also provides a method as described herein, wherein the one or more cytokines are selected from the group consisting of ifnγ, TNF, GMCSF, MIG (CXCL 9), IP-10 (CXCL 10), and MCP1 (CCL 2).

The invention also provides a method as described herein, wherein the activation is measured as an increase in direct killing of target cells.

In some embodiments, the method further comprises administering a second antibody.

In some embodiments, the second antibody is an antibody that binds to and/or inhibits a human checkpoint receptor protein.

In some embodiments, the second antibody is selected from the group consisting of: anti-PVRIG antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-CTLA-4 antibodies, anti-PD-L2 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-CEACAM-1 antibodies, anti-PVR antibodies, anti-LAG 3 antibodies, anti-CD 112 antibodies, anti-CD 96 antibodies, anti-TIM 3 antibodies, anti-BTLA antibodies, anti-ICOS antibodies, anti-OX 40 antibodies or anti-41 BB antibodies, anti-CD 27 antibodies or anti-GITR antibodies.

In some embodiments, the PVRIG antibody is selected from the group consisting of cha.7.518.1.h4 (S241P) and cha.7.538.1.2.h4 (S241P).

In some embodiments, the anti-PVRIG antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CHA.7.518.1.H4 (S241P) (SEQ ID NO: 260) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CHA.7.518.1.H4 (S241P) (SEQ ID NO: 265).

In some embodiments, the anti-PVRIG antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CHA.7.538.1.2.H4 (S241P) (SEQ ID NO: 270) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CHA.7.538.1.2.H4 (S241P) (SEQ ID NO: 275).

In some embodiments, the anti-PVRIG antibody comprises: i) Including those from CHA.7.518.4 (SEQ ID NO:1453; FIG. 36 AG) the heavy chain variable domains vhCDR, vhCDR2, and vhCDR3 and ii) the heavy chain variable domain comprising a heavy chain variable domain from CHA.7.518.4 (SEQ ID NO:1457; fig. 36 AG) of the vlCDR, vlCDR, and vlCDR 3.

In some embodiments, the anti-PVRIG antibody is selected from the group consisting of: GSK4381562/SRF816 (GSK/Surface), NTX2R13 (Nectin Therapeutics), anti-PVRIG antibodies as described in WO 2017/04004, anti-PVRIG antibodies as described in WO 2001/008879, anti-PVRIG antibodies as described in WO 2018/017864 and anti-PVRIG antibodies as described in WO 2118/000205.

In some embodiments, the anti-TIGIT antibody is selected from the group consisting of cpa.9.083.h4 (S241P) and cpa.9.086.h4 (S241P).

In some embodiments, the anti-TIGIT antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2 and vhCDR3 from CPA.9.083.H4 (S241P) (SEQ ID NO: 350) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2 and vlCDR3 from CPA.9.083.H4 (S241P) (SEQ ID NO: 355).

In some embodiments, the anti-TIGIT antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2 and vhCDR3 from CPA.9.086.H4 (S241P) (SEQ ID NO: 360) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2 and vlCDR3 from CPA.9.086.H4 (S241P) (SEQ ID NO: 365).

In some embodiments, the anti-TIGIT antibody comprises: i) Including those from CHA.9.547.18 (SEQ ID NO:1177; FIG. 34 QQQQ) the heavy chain variable domains of vhCDR1, vhCDR2 and vhCDR3 and ii) a polypeptide comprising a polypeptide derived from CHA.9.547.18 (SEQ ID NO:1181; fig. 34 QQQQ) of the light chain variable domains vlCDR, vlCDR2 and vlCDR 3.

In some embodiments, the anti-TIGIT antibody is selected from the group consisting of: EOS-448 (GlaxoSmithKline, iTeos Therapeutics), BMS-986207, duwanalimumab (AB 154, arcus Biosciences, inc.), AB308 (Arcus Bioscience), european-amber Li Shan antibody (aBGB-A1217, beiGene), tiarey Li Youshan antibody (MTIG7192A,RocheGenentech)、BAT6021(Bio-Thera Solutions)、BAT6005(Bio-Thera Solutions)、IBI939(Innovent Biologics,US2021/00040201)、JS006(Junshi Bioscience/COHERUS)、ASP8374(Astellas Pharma Inc)、 vitamin-Bo Li Shan antibody (MK-7684,Merck Sharp&Dohme), M6332 (MERCK KGAA), ai Tili mab (OMP-313M32,Mereo BioPharma)、SEA-TGT(Seagen)y、HB0030(Huabo Biopharma)、AK127(AKESO)、IBI939(Innovent Biologics), and anti-TIGIT antibodies include Genntech antibodies (MTIG 7192A).

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: nawuzumab @BMS; CHECKMATE 078) and pembrolizumabMerck), TSR-042 (Tesaro), cimipn Li Shan anti (REGN 2810; regeneron Pharmaceuticals, see US 20170174779), BMS-936559, swadazumab (PDR 001, novartis), dermatitid (CT-011; pfizer Inc.), tirelimumab (BGB-A317, beiGene), carilimumab (SHR-1210, incyte and Jiangsu HengRui), SHR-1210 (CTR 20170299 and CTR 20170322), SHR-1210 (CTR 20160175 and CTR 20170090), xindi Li Shan antibodyElily and Innovent Biologics), terlipressin Li Shan antibody (JS 001, shanghai Junshi Bioscience), JS-001 (CTR 20160274), IBI308 (CTR 20160735), BGB-a317 (CTR 20160872), pi An Puli mab (AK 105, akeso Biopharma), sirolimus (Arcus), BAT1306 (Bio-Thera Solutions Ltd), saran Li Shan antibody (PF-06809591, pfizer), doralimus (Dostarlimab-gxly) (GlaxoSmithKline LLC), palo Li Shan antibody (Biocad), california Li Shan antibody (Akeso Inc), jerland Li Shan antibody (Genor BioPharma Co Ltd), s Lu Lishan antibody (Shanghai Henlius Biotech Inc), baterimumab (Shanghai Henlius Biotech Inc), refform Shanghai Henlius Biotech Inc antibody (Incyte Corp), cerilimumab (Johnson & Johnson), CS-1003 (Shanghai Henlius Biotech Inc), IBI-318 (Shanghai Henlius Biotech Inc), exemestane (Shanghai Henlius Biotech Inc), pralidoxime antibody (Shanghai Henlius Biotech Inc), QL-1604 (Shanghai Henlius Biotech Inc), shanghai Henlius Biotech Inc-10 2, terpolizumab (Shanghai Henlius Biotech Inc), principal d-7789 (astraneca) and Shanghai Henlius Biotech Inc antibodies (Shanghai Henlius Biotech Inc), EMB-02 (Shanghai Henlius Biotech Inc), ellizumab (Shanghai Henlius Biotech Inc), ependab (Shanghai Henlius Biotech Inc), rufive3932, shanghai Henlius Biotech Inc, and rufig3932 (Shanghai Henlius Biotech Inc) antibodies (Shanghai Henlius Biotech Inc) YBL-006 (Y-Biologics Inc) and ONO-4635 (Ono Pharmaceutical Co Ltd), LY-3434172 (ELI LILLY AND Co).

In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: alemtuzumab @MPDL3280A; IMpower110,110; roche/Genentech, avermectinMSB001071 8C; EMD serrono & Pfizer) and dewaruzumab (MEDI 4736; AstraZeneca). And other antibodies being developed, such as lodalimab (LY 3300054, eli Lily), pi Weishan anti (Jounce Therapeutics Inc), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), en Wo Lishan anti (Jiangsu Simcere Pharmaceutical Co Ltd), shu Geli mab (CStone Pharmaceuticals Co Ltd), ke Xili mab (Checkpoint Therapeutics Inc), parker Mi Lishan anti (CytomX Therapeutics Inc)、IBI-318、IBI-322、IBI-323(Innovent Biologics Inc)、INBRX-105(Inhibrx Inc)、KN-046(Alphamab Oncology)、6MW-3211(Mabwell Shanghai Bioscience Co Ltd)、BNT-311(BioNTech SE)、FS-118(F-star Therapeutics Inc)、GNC-038(Systimmune Inc)、GR-1405(Genrix(Shanghai)Biopharmaceutical Co Ltd)、HS-636(Zhejiang Hisun Pharmaceutical Co Ltd)、LP-002(Lepu Biopharma Co Ltd)、PM-1003(Biotheus Inc)、PM-8001(Biotheus Inc)、STIA-1015(ImmuneOncia Therapeutics LLC)、ATG-101(Antengene Corp Ltd)、BJ-005(BJ Bioscience Inc)、CDX-527(Celldex Therapeutics Inc)、GNC-035(Systimmune Inc)、GNC-039(Systimmune Inc)、HLX-20(Shanghai Henlius Biotech Inc)、JS-003(Shanghai Junshi Bioscience Co Ltd)、LY-3434172(Eli Lilly and Co)、MCLA-145(Merus NV)、MSB-2311(Transcenta Holding Ltd)、PF-07257876(Pfizer Inc)、Q-1802(QureBio Ltd)、QL-301(QLSF Biotherapeutics Inc)、QLF-31907(Qilu Pharmaceutical Co Ltd)、RC-98(RemeGen Co Ltd)、TST-005(Transcenta Holding Ltd)、 alemtuzumab (IMpower 133), BMS-936559/MDX-1105 and/or RG-7446/MPDL3280A, and YW243.55.S70.

In some embodiments, the anti-IL 18-BP antibody and the second antibody are administered sequentially or simultaneously in any order and in one or more formulations.

In some embodiments, the anti-IL 18-BP antibody is for use in combination with an immunostimulatory antibody, cytokine therapy or immunomodulatory drug, cytotoxic agent, chemotherapeutic agent, growth inhibitory agent, anti-hormonal agent, kinase inhibitor, anti-angiogenic agent, cardioprotective agent, immunosuppressant, agent that promotes blood cell proliferation, angiogenesis inhibitor, protein Tyrosine Kinase (PTK) inhibitor, or other therapeutic agent.

In some embodiments, the method further comprises administering one or more inflammatory body activators.

In some embodiments, the inflammatory body activator is a chemotherapeutic agent.

In some embodiments, the chemotherapeutic agent is selected from the group consisting of platinum, paclitaxel (taxol), sorafenib, doxorubicin, sorafenib, 5-FU, gemcitabine, and irinotecan (CPT-11).

In some embodiments, the platinum chemotherapeutic agent is oxaliplatin or cisplatin.

In some embodiments, the inflammatory body activator is a CD39 inhibitor.

In some embodiments, the CD39 inhibitor is an anti-CD 39 antibody.

In some embodiments, the cancer is selected from the group consisting of: vascularized tumors, melanomas, non-melanoma skin cancers (squamous cell carcinoma and basal cell carcinoma), mesotheliomas, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine lung cancer (including pleural mesothelioma, neuroendocrine lung cancer), NSCL (large cell), NSCLC large cell adenocarcinoma, non-small cell lung cancer (NSCLC), NSCLC squamous cell carcinoma, soft tissue sarcoma, kaposi's sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, PDL1> = NSCLC of 50% TPS, neuroendocrine lung cancer, atypical carcinoid lung cancer, peritoneal carcinoma, esophageal carcinoma, hepatocellular carcinoma, liver cancer (including HCC), gastric cancer (GASTRIC CANCER), gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, urothelial carcinoma, bladder cancer, liver cancer, glioma, brain cancer (and oedema, such as oedema associated with brain tumor), breast cancer (including, for example, triple negative breast cancer), testicular cancer, testicular germ cell carcinoma, colon cancer, colorectal cancer (CRC), colorectal cancer (MSS-MSS); refractory MSS colorectal cancer; MSS (microsatellite steady state), primary peritoneal carcinoma, primary peritoneal ovarian carcinoma, microsatellite stabilized primary peritoneal carcinoma, platinum resistant microsatellite stabilized primary peritoneal carcinoma, CRC (MSS unknown), rectal carcinoma, endometrial carcinoma (endometrial cancer) (including endometrial carcinoma (endometrial carcinoma)), uterine carcinoma, salivary gland carcinoma, renal cell carcinoma (RENAL CELL CANCER, RCC), renal cell carcinoma (RENAL CELL carnoma, RCC), gastroesophageal junction carcinoma, prostate carcinoma, vulval carcinoma, thyroid carcinoma, liver cancer, carcinoid, head and neck carcinoma, B cell lymphoma (including non-Hodgkin's lymphoma), low-grade/follicular non-Hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, diffuse large B cell lymphoma, high-grade immunoblast NHL, high-grade lymphoblast NHL, high-grade nuclear cell lymphoma, megawatt lymphomatoid, and AIDS-related lymphomasMacroglobulinemia), hodgkin's lymphoma (HD), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute Myelogenous Leukemia (AML), hairy cell leukemia, chronic myeloblastic leukemia, multiple myeloma, post-transplant lymphoproliferative disease (PTLD), abnormal vascular proliferation associated with mole-type hamartoma, meigs' syndrome, merkel cell carcinoma, high MSI carcinoma, KRAS mutant tumors, adult T-cell leukemia/lymphoma, adenoid cystic carcinoma (including adenoid cystic carcinoma), melanoma, malignant melanoma, metastatic melanoma, pancreatic carcinoma, ovarian carcinoma (ovarian cancer) (including ovarian carcinoma (ovarian carcinoma)), pleural mesothelioma, cervical squamous cell carcinoma (neck SCC), anal SCC), non-primary carcinoma, uterine carcinoma, pleural mesothelioma, endometrial carcinoma, chondrosarcoma, endometrial sarcoma, astrocytoma, fibroid carcinoma, and Anaplastic (AL), and Amyloidosis (AL).

In some embodiments, the cancer is selected from the group consisting of: clear cell carcinoma (RCC), lung cancer, NSCLC, lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple Negative Breast Cancer (TNBC), head and neck tumors, colorectal adenocarcinoma, melanoma, and metastatic melanoma.

The invention also provides anti-IL 18BP antibodies as described herein for use in treating cancer by activating T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells and/or congenital lymphoid cells (ILCs) and/or modulating bone marrow cells of a patient.

The invention also provides anti-IL 18BP antibodies as described herein for increasing IL-18 mediated immunostimulatory activity in the Tumor Microenvironment (TME) and/or lymph nodes.

The invention also provides anti-IL 18BP antibodies as described herein for use in treating cancer in a recipient patient.

The invention also provides an anti-IL 18BP antibody as described herein for use according to any one of the preceding claims.

The invention also provides for the use of an anti-IL 18BP antibody in combination with a second antibody as described herein. In some embodiments, the second antibody is selected from the group consisting of an anti-PVRIG antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-TIGIT antibody.

The invention also provides an anti-IL 18BP antibody as described herein, wherein the anti-IL 18BP antibody exhibits a binding affinity or KD of less than 0.005pM、0.01pM、0.02pM、0.03pM、0.04pM、0.05pM、0.06pM、0.07pM、0.08pM、0.09pM、0.10pM、0.15pM、0.20pM、0.25pM、0.30pM、0.35pM、0.40pM、0.45pM、0.50pM、0.55pM、0.60pM、0.65pM、0.70pM、0.75pM、0.80pM、0.85pM、0.90pM、0.95pM or 1 pM.

Drawings

Fig. 1A to 1L depict vhCDR, vhCDR2, vhCDR3, vlCDR1, vlCDR2, vlCDR3 sequences of antibodies 66650 (fig. 1A and 1E and 1I), 66670 (fig. 1B and 1F and 1J), 66692 (fig. 1C and 1G and 1K), 66716 (fig. 1D and 1H and 1L). FIG. 1M provides IgG sequences, including IgG1, igG2, igG3, and IgG4.

Fig. 2A-2U depict the full length of variable heavy and light chains, vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, vlCDR3 sequences and the following antibodies: ADI-71709 (FIG. 2A), ADI-71719 (FIG. 2B), ADI-71720 (FIG. 2C), ADI-71722 (FIG. 2D), ADI-71701 (FIG. 2E), ADI-71663 (FIG. 2F), ADI-71662 (FIG. 2G), ADI-66692 (FIG. 2H), ADI-71710 (FIG. 2I), ADI-71717 (FIG. 2J), ADI-71739 (FIG. 2K), ADI-71736 (FIG. 2L), ADI-71707 (FIG. 2M), ADI-66716 (FIG. 2N), ADI-71728 (FIG. 2O), ADI-71741 (FIG. 2P), ADI-71742 (FIG. 2Q), ADI-71744 (FIG. 2R), ADI-71753 (FIG. 2S), ADI-71755 (FIG. 2T) and AB-837 (also referred to as "AbD35328", "837" or "837") (FIG. 2U).

Fig. 3A to 3E: a) Alignment of CDRH and CDRL sequences between VH3-23 and VL-kappa-1-12 germline sequences 71663 and 71662 and 66692 is depicted. B) Alignment of CDRH and CDRL sequences between VH1-03 and VL-kappa-1-5 germline sequences 71701, 71707, 71709, 71710 and 71717 is depicted. C) Alignment of CDRH and CDRL sequences between VH1-69 and VL-kappa-1-2 germline sequences 71719, 71720, 71722 and 71728 is depicted. D) Alignment of CDRH and CDRL sequences between VH4-39 and VL-kappa-1-12 germline sequences 71736, 71739 and 66716 is depicted. E) Alignment of CDRH and CDRL sequences between VH4-39 and VL-kappa-1-12 germline sequences 71736, 71739, 66716, 71742, 71744, 71741, 71753 and 71755 is depicted.

Fig. 4A to 4B: a) Depicts the expression of IL18 in all TCGA tumors, and B) depicts the expression of IL18-BP in all TCGA tumors. A plot of log10RPKM for each TCGA tumor, reference line at 1 RPKM.

Fig. 5A to 5B: a) IL18 stratified by ifnγ expression for each tumor type in TCGA is depicted. B) IL18-BP stratified by IFNγ expression for each tumor type in TCGA is depicted. A plot of log10 RPKM for each TCGA tumor, reference line at 1 RPKM. For tumor abbreviations, see table 1.Ifnγ high represents the upper quartile and ifnγ low represents the lower quartile. FC-fold change, P-student T test P-value between ifnγ high and ifnγ low. The fraction represents the number of samples with high/low ifnγ.

Fig. 6A to 6B: a) Core inflammatory body signatures stratified by ifnγ expression for each tumor type in TCGA are depicted. A plot of log10 RPKM for each TCGA tumor, reference line at 1 RPKM. For tumor abbreviations, see table 1.Ifnγ high represents the upper quartile and ifnγ low represents the lower quartile. FC-fold change, P-student T test P-value between ifnγ high and ifnγ low. The fraction represents the number of samples with high/low ifnγ. B) A cosine similarity heat map and a dendrogram between the core inflammatory minibody gene, IL18-BP, IL18R gene and the additional upstream inflammatory minibody gene are depicted.

Fig. 7A to 7B: a) A dot plot of IL18 and IL18-BP in breast cancer subtypes before and at treatment is depicted, demonstrating expression of both genes before and at TNBC treatment. B) A dot plot of IL18 and IL18-BP is depicted, demonstrating the expression of these two genes prior to and at the time of TNBC treatment, also divided by extended TCR clone (_e) and non-extended TCR clone (_ne).

Fig. 8: affinity matrix of mAbs against human IL18-BP obtained by Biacore for human and cynomolgus monkey ("cyno") IL18-BP

Fig. 9: IL18-BP-Fc was competitively bound to human IL18 in AlfaLISA assays using purified Ab at 15nM and hIgG1 backbone.

Fig. 10: blocking Activity of parent mAb against human IL18-BP by ELISA

Fig. 11: blocking Activity of parent mAbs against cynomolgus monkey IL18-BP by ELISA

Fig. 12: IC50 values of anti-human IL18-BP Ab measured by ELISA

Fig. 13: the ability of IL18-BP-Fc protein to bind human IL18 was rescued using mAbs against human IL18-BP demonstrated by IL18 HEK293 reporter cells.

Fig. 14A to 14H: the anti-IL-18 BP antibody fully restored IL-18 activity on NK cells. FIGS. 14A and 14H show schematic diagrams of assay settings; thawed NK cells from four donors were incubated with rhIL-18 (3 ng/ml or 10 ng/ml) and rhIL-18BP (1. Mu.g/ml) in the presence of rhIL-12 (10 ng/ml) for 30 min to form IL-18-IL-18BP complex. After 30 minutes of incubation, the cells were treated by dose titration of anti-IL-18 BP antibody (20. Mu.g/ml to 0.25. Mu.g/ml; dilution factor 1:3 (FIGS. 14A to 14G), or 10. Mu.g/ml to 0.325. Mu.g/ml; dilution factor 1:2 (FIGS. 14H to 14N)) or isotype control (20. Mu.g/ml (FIGS. 14A to 14G) or 10. Mu.g/ml (FIGS. 14A to 14G)). Fig. 14I-14N show that anti-IL-18 BP antibodies were able to fully restore ifnγ secretion (fig. 14B-14D, 14I-14N) and CD69 expression (fig. 14E-14G) in a dose-dependent manner. Isotype control failed to restore IL-18 activity. FIG. 14N shows the% dose response curve and calculated EC50 for rescue of anti-IL-18 BP antibody. Representative data is from one donor. Rescue of anti-IL-18 BP Ab was calculated as: [ (IL-12+IL-18+IL-18 BP+anti-IL-18 BP Ab) - (IL-12+IL-18+IL-18 BP+ isoform) ]/[ (IL-12+IL-18) - (IL-12+IL-18+IL-18 BP+ isoform) ].

Fig. 15A to 15J: anti-IL-18 BP antibodies block IL-18BP secreted by PBMC. FIGS. 15A and 15D are schematic diagrams showing measurement settings; thawed PBMC from both donors were incubated with rhIL-12 (10 ng/ml), rhIL-18 (33.3 ng/ml) and dose-titrated anti-IL-18 BP antibody (FIG. 15B: 20. Mu.g/ml to 0.625. Mu.g/ml; dilution factor 1:2. FIG. 15E to 15J: 6. Mu.g/ml to 0.002. Mu.g/ml; dilution factor 1: 3) or isotype control (20. Mu.g/ml) for 24 hours. FIGS. 15B-15C and 15E-15J show that anti-IL-18 BP antibodies were able to induce dose-dependent IFN gamma secretion above IL-12+IL-18 control levels, indicating that the antibodies may block endogenous IL-18BP activity. Representative data is from one donor.

FIG. 16 depicts affinity measurements of anti-mouse mIL18BP Ab to mouse IL18-BP protein obtained by ELISA.

FIG. 17 depicts SPR kinetic measurements of anti-mouse IL18-BP (AbD 35328 (also referred to as "837", "Ab837" or "AB-837")).

FIG. 18 depicts the performance of mAbs in functional blockade of mIL18-BP-mIL-18 interactions by ELISA.

FIG. 19 depicts an IC50 analysis of anti-mouse IL18-BP (AbD 35328).

FIG. 20 depicts the functional blocking activity of purified mAbs on mouse IL18-BP by IFN gamma secretion.

FIG. 21 depicts an EC50 analysis against mouse IL 18-BP.

FIGS. 22A-22L depict evaluation of anti-IL 18-BP monotherapy or combination therapy with anti-PD-L1 Ab in a mouse syngeneic CT26 tumor model. (A): tumor growth measurement for each group in monotherapy, (B): percent survival analysis for each group in monotherapy, (C) - (F): summary of tumor growth measurements for each individual group of mice in monotherapy, (G): tumor growth measurement for each group in combination therapy, (H): percent survival analysis for combination therapies, (I) - (K): tumor growth measurement summary for each individual group of mice in combination therapy, and (L): statistical analysis of the effect of combination therapies.

FIGS. 23A-23L depict evaluation of anti-IL 18-BP monotherapy or combination therapy with anti-PD-L1 Ab in a mouse isogenic B16/Db-hmgp100 mouse tumor model. (A): tumor growth measurement for each group in monotherapy, (B): percent survival analysis for each group in monotherapy, (C) - (F): summary of tumor growth measurements for each individual group of mice in monotherapy, (G): tumor growth measurement for each group in combination therapy, (H): percent survival analysis for each group in combination therapy, (I) - (K): tumor growth measurement summary for each individual group of mice in combination therapy, and (L): statistical analysis of the effect of combination therapies.

FIGS. 24A-24G depict the activity of anti-IL 18-BP and anti-TIGIT combinations in B16/Db-hmgp100 syngeneic mouse tumor models. (A): tumor growth measurement for each group in combination therapy, (B): percent survival analysis for each group of combination therapies, (C) - (F): tumor growth measurement summary of individual mice in each group of combination therapy, and (G): statistical analysis of the effect of combination therapies.

FIGS. 25A-25G depict the activity of anti-IL 18-BP and anti-PVRIG combinations in B16/Db-hmgp100 syngeneic mouse tumor models. (A): tumor growth measurement for each group in combination therapy, (B): percent survival analysis for each group in combination therapy, (C) - (F): tumor growth measurement summary of individual mice in each group of combination therapy, and (G): statistical analysis of the effect of combination therapies.

FIGS. 26A-26G depict anti-IL 18-BP and anti-mPD-L1 monotherapy activity in an isogenic E0771 in situ mouse tumor model. (A): tumor growth measurements for each group in monotherapy, (B) - (E): summary of tumor growth measurements for each individual group of mice in monotherapy, (F): survival analysis for each group in monotherapy, and (G): statistical analysis of monotherapy effects.

Fig. 27A-27F depict tumor re-challenge experiments for the E0771 TNBC model. A group of 5 to 10C 57BL/6 tumor age-matched naive mice (0.5X10 ⁶ cells) was vaccinated in situ with E0771. When the tumor reached a volume of 250mm ³, mice were treated with the indicated mabs: AB-837mIgG1-D265A or isotype control followed by 5 additional doses. Two months later, tumor-free and age-matched naive mice were re-vaccinated in situ with E0771. (A): tumor volumes are expressed as mean volume±sem. (B) Separate tumor measurements, CR-complete responders, PR-partial responders (TV < = 500mm ³) for each mouse are depicted. (C) shows Kaplan-Meier survival curves for each group. (D) spleen weight/body weight ratio. (E) Percentage of CD44 ⁺CD62L^-CD8⁺ effector T cells. (F) number of CD19+ cells per mg spleen.

FIGS. 28A-28F depict the amino acid sequences of human (A) and mouse (C) IL18-BP proteins. The signal peptide sequence is highlighted. Secreted human and mouse IL18-BP protein chains are depicted in (B) and (D), respectively. FIGS. 28E and 28F depict the amino acid sequences of human and mouse IL18 proteins, respectively.

Fig. 29A-29B depict the variable heavy and light chains of cha.7.518.1.h4 (S241P) and cha.7.538.1.2.h4 (S241P) sequences vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR 3.

Fig. 30A to 30B depict the variable heavy and light chains of cpa.9.083.h4 (S241P) and cpa.9.086.h4 (S241P) and vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR sequences.

FIG. 31 shows the ability of mAbs against human IL18-BP to rescue IL18-BP binding to human IL18 in human serum demonstrated by ELISA

FIG. 32 shows the ability of mAbs against human IL18-BP to rescue cyno IL18-BP binding to cyno IL18 demonstrated using ELISA.

FIG. 33 shows that TIGIT and IL18Ra are co-expressed in TME.

Fig. 34A-34 QQQQ depict the sequences of four anti-TIGIT antibodies (cpa.9.083.h4 (S241P), cpa.9.086.h4 (S241P), cha.9.547.7.h4 (S241P) and cha.9.547.13.h4 (S241P)) as well as reference antibodies (BM 26 and BM 29) and many other anti-TIGIT antibodies that block TIGIT and PVR interactions.

Fig. 35A-35B depict the amino acid sequences of human IgG1 (with some useful amino acid substitutions), igG2, igG3, igG4, constant domains of IgG4 with hinge variants particularly useful in the present invention, and constant domains of kappa and lambda light chains.

FIGS. 36A-36 AG depict the variable heavy and light chain and vhCDR, vhCDR2, vhCDR3, vlCDR1, vlCDR and vlCDR3 sequences of an anti-PVRIG antibody of the invention.

Fig. 37A to 37D depict sequences of other PVRIG antibodies of the present invention.

FIGS. 38A-38X provide additional anti-PVRIG antibodies for use in the present invention.

Fig. 39A-39B depict sequences of exemplary anti-PD-1 antibodies.

Fig. 40A-40I depict sequences of exemplary anti-PD-L1 antibodies.

FIGS. 41A-41D depict Biacore KD measurements with biotinylated human/cyno IL18BP-Fc protein coated on CM5 chips. Fig. 41 (a), (B): biacore images of anti-IL 18BP Fab-human IL18BP interactions; 10min dissociation (41 (A)), 85 min dissociation (41 (B)). Fig. 41 (C), (D): biacore images of anti-IL 18BP Fab-cyno IL18BP interactions, 10min dissociation (41 (C)), 85 min dissociation (41 (D)).

FIG. 42 depicts a table showing KD values for human/cyno anti-IL 18BP Fab-IL18BP interactions measured by Biacore.

Fig. 43A-43B present the affinity of the optimized IL18BP antibodies evaluated using MSD. FIG. 43A shows the superposition of a Fab-IL18BP MSD image (black) with a human IL-18-IL18BP MSD image (green). FIG. 43B shows the superposition of a Fab-IL18BP MSD image (black) with a Cyno IL-18-IL18BP MSD image (green).

FIG. 44 presents a table showing KD values for human/cyno anti-IL 18BP Fab-IL18BP interactions as measured by MSD.

FIG. 45 presents a table showing KD values for human/cyno IL18-IL18BP interactions as measured by MSD.

FIG. 46 provides exemplary antibody features of an αIL-18BP antibody of interest (αIL-18BP Ab).

FIG. 47 shows elevated IL-18BP levels in human cancers. Expression of IL18BP transcripts in normal (green) or cancer (red) tissues from TCGA database. GBM, glioblastoma multiforme; HSNC squamous cell carcinoma of head and neck; KIRC, renal clear cell carcinoma; PAAD, pancreatic cancer; SKCM, cutaneous melanoma; STAD, gastric adenocarcinoma (< P0.01).

FIG. 48 shows the expression of IL-18BP in an inhibitory myeloid cell population of TME, indicating a resistance mechanism. Single cell RNA analysis of tumor infiltrating bone marrow cells, including Tumor Associated Macrophages (TAMs) and Dendritic Cells (DCs) in human colorectal cancer (CRC), showed expression of IL-18BP in an inhibitory bone marrow cell population in tumors. This suggests a mechanism of resistance to immune activation in the Tumor Microenvironment (TME). Left diagram: peripheral (PBMCs), normal tumors (NAT) and bone marrow cell populations in tumors. Right figure: IL18BP is expressed predominantly in the cDC2-CD1C and TAM-C1 QC-inhibited myeloid cell populations, suggesting that IL18BP may be a resistance mechanism to immune cell activation in tumors.

FIGS. 49A-49B provide that αIL-18BP Ab (ADI-71739) enhances stimulatory activity of human T cells. A) Schematic of the assay setup; thawed Tumor Infiltrating Lymphocytes (TILs) co-cultured with MEL624 cells at a 1:1 ratio were treated with rhIL-18 (R & D system, 30 ng/ml) and rhIL-18BP (R & D system, 1 μg/ml) for 30 minutes to form IL-18-IL-18BP complex. After 30 minutes incubation, cells were treated with ADI-71739 or isotype control (10 ug/ml). B) It was shown that the anti-IL-18 BP antibodies were able to increase ifnγ secretion from TIL compared to isotype control. C) Schematic of the assay setup; MEL-624 cells overexpressing PD-L1 were loaded with CMV pp65 peptide and inoculated. Cells were incubated with rhIL-18 (30 ng/ml) and rhIL-18BP (2. Mu.g/ml) for 30 minutes to form IL-18-IL-18BP complex. Cells were then treated with ADI-71739, pembrolizumab or isotype control (all antibodies were administered at the same final concentration of 10 μg/ml). After 30 minutes incubation with the antibody, CMV-reactive T cells were added to the culture. D) ADI-71739 as monotherapy is capable of increasing IFN gamma secretion from CMV-reactive T cells and secretion in a more efficient manner when combined with pembrolizumab.

FIGS. 50A-50B show that anti-IL-18 BP antibodies fully restored IL-18 activity in the MEL624:TIL assay. Fig. 50A: schematic of the assay setup; thawed Tumor Infiltrating Lymphocytes (TILs) co-cultured with MEL624 cells at a 1:1 ratio were treated with rhIL-18 (30 ng/ml) and rhIL-18BP (1 μg/ml) for 30 minutes to form IL-18-IL-18BP complex. After 30 minutes of incubation, anti-IL-18 BP antibody (ADI-71722) (30. Mu.g/ml to 0.01. Mu.g/ml; dilution factor 1:3) or isotype control (30. Mu.g/ml) was incubated together for 24 hours by dose titration. Fig. 50B: it was shown that anti-IL-18 BP antibodies were able to fully resume ifnγ secretion in a dose-dependent manner. Isotype control failed to restore IL-18 activity. Fig. 50C: the% dose response curve and calculated EC50 for rescue of anti-IL-18 BP antibodies are shown. Representative data is from one donor. Rescue of anti-IL-18 BP Ab was calculated as: [ (IL-18+IL-18 BP+anti-IL-18 BP Ab) - (IL-18+IL-18 BP+ isoform) ]/[ (IL-18) - (IL-18+IL-18 BP+ isoform) ].

FIGS. 51A-51B show that anti-hIL-18 BP antibodies enhance PD-1 and DNAM-1 axis blocking activity in an in vitro CMV recall assay. anti-IL-18 BP antibody increased CMV-reactive T cell secretion IFNg both as monotherapy and in combination with aPVRIG/aTIGIT/pembrolizumab. A) Schematic of the assay setup; MEL-624 cells overexpressing PD-L1 were loaded with CMV pp65 peptide and inoculated. Cells were incubated with rhIL-18 (30 ng/ml) and rhIL-18BP (2. Mu.g/ml) for 30 minutes to form IL-18-IL-18BP complex. Cells were then treated with anti-IL-18 BP antibody (ADI-71722), anti-PVRIG, anti-TIGIT, pembrolizumab or isotype control (all antibodies were administered at the same final concentration of 10. Mu.g/ml). After 30 minutes incubation with the antibody, CMV-reactive T cells were added to the culture. B) ADI-71722 increased secretion of IFNγ by CMV-reactive T cells both as monotherapy and in combination with anti-PVRIG/anti-TIGIT/pembrolizumab. Left diagram: anti-IL-18 BP antibodies were able to fully restore ifnγ secretion as monotherapy and were secreted in a more efficient manner when combined with pembrolizumab/anti-PVRIG. Right figure: ADI-71722 was able to fully restore IFNγ secretion as monotherapy and was secreted in a more efficient manner when combined with pembrolizumab/anti-TIGIT.

The data provided in FIG. 52 shows that ADI-71739 binds human and cyno IL-18BP with high affinity and mouse IL-18BP with low affinity. ADI-71739 binds human and cyno IL-18BP with high affinity and mouse IL-18BP with low affinity: upper graph (left to right): human IL18-IL18BP interaction measurement, cyno IL18-IL18BP interaction measurement and mouse IL18-IL18BP interaction measurement in KinExA had final KD of 441fM, 345fM and 3.7pM, respectively. The following graph (left to right): ADI-71739-human IL18BP interaction measurement, ADI-71739-cyno IL18BP interaction measurement in KinExA, and ADI-71739-mouse IL18BP interaction measurement obtained by Biacore. The final KD was 291fM, 209fM and 4nM, respectively. For each run in KinExA, two or three curves with different Column Binding Protein (CBP) concentrations were run and analyzed using an n-curve analysis to determine Kd.

FIG. 53 shows the blocking effect of anti-IL 18BP Ab on human IL18BP binding to human IL-18. The blocking effect of anti-IL 18BP Ab was tested by ELISA using 1ng/ml human IL-18 protein.

FIG. 54 shows a competition ELISA using complexes of soluble IL18-IL18BP and anti-IL 18BP Ab. Blocking of IL18-IL18BP complex formation was tested by ELISA, and MAB1191 showed reduced blocking activity compared to 66716 Ab.

Fig. 55 shows that IL18Ra is expressed on a sub-population of TIL of TME and is induced to be expressed on TIL compared to the periphery. IL18Ra is expressed on the TIL of TME and induces its expression on CD4 TIL compared to the periphery. A) The expression of IL18Ra on cd8+ and cd4+ and NK TIL from dissociated human tumors of various cancer types is shown. Each point represents a different tumor of an individual patient. Fold expression values were calculated by dividing the MFI of the target by the MFI of the relevant isotype control. (FOI). Mean and SEM are shown by scale. B) Expression of IL18Ra on cd4+ and cd8+ T cells and NK cells from donor matched PBMC and TMEs. Statistical analysis using paired t-test (two-tailed), P <0.05; * P=0.0064

Figure 56 shows that IL18 levels in serum of cancer patients are increased compared to levels in HD serum. A) Levels of IL18 analytes (IL 18 and IL18 BP) in serum of patients of different indications. B) A dot plot representing IL18 analyte in serum samples from individual patients or HD. Statistical analysis using t-test (double tail) with p.ltoreq.0.0005%

Fig. 57 shows IL18 analyte (IL 18 and IL18 BP) levels in tumor-derived supernatants (TDS) for different indications. A plot of IL18 analyte in a TDS sample is shown. Each dot represents a separate patient sample.

Fig. 58A-58B show the levels of IL18 (a) and IL18BP (B) in tumor-derived supernatants (TDS) of patients of different indications. Average levels are indicated by black lines.

FIGS. 59A-59C show the expression of IL-18BP in an inhibitory myeloid cell population of TME, indicating a resistance mechanism. IL-18BP is expressed in an inhibitory myeloid cell population and is associated with PD-L1 in TME, indicating a resistance mechanism. A) In colon and breast cancers, IL-18BP is associated with PD-L1 at the RNA level (TCGA), which suggests a mechanism of resistance to immune activation in the Tumor Microenvironment (TME). B) Single cell RNA analysis of tumor infiltrating bone marrow cells, including tumor-associated macrophages (TAMs) and Dendritic Cells (DCs) in colon cancer patients, showed up-regulation of IL-18BP in bone marrow cell populations in TME compared to Peripheral (PBMC), indicating a resistance mechanism to immune activation in TME. C) Single cell RNA analysis of tumor infiltrating bone marrow cells, including tumor-associated macrophages (TAMs) and Dendritic Cells (DCs) in different indications, showed up-regulation of IL-18BP in bone marrow cell populations in TME compared to Peripheral (PBMC), suggesting a resistance mechanism to immune activation in TME

FIGS. 60A-60D show IL-18BP upregulation following Immune Checkpoint Blocking (ICB) treatment. A) To C) IL-18BP up-regulation (RNA levels) after ICB treatment, IL-18BP levels up-Regulated (RNA) in the tumor microenvironment after treatment with anti-PD-1 (breast and basal cell carcinoma) or anti-PD-1 plus anti-CTLA-4 (melanoma), indicating a potential resistance mechanism. D) The rise in IL-18BP in serum of NSCLC patients following treatment with agd- (L) 1 quantifies plasma IL-18BP protein levels at baseline in healthy donors (n=22) and NSCLC patients (n=52) prior to treatment and after receiving treatment with anti-PD- (L) 1 (n=52) by ELISA.

FIGS. 61A-61B show that IL-18BP baseline serum levels may be correlated with poor responses against PD-1. IL-18BP is supportive data of soluble ICP and the action of potential resistance mechanisms to PD1 blockade in renal cell carcinoma patients receiving Pem Shan Kangjia lenvatinib. A) High IL-18BP in serum of patients pre-treated with pembroliquanib Shan Kangjia was associated with a shorter Progression Free Survival (PFS). B) High IL-18BP associated with stable or progressive disease (SD/PD) in patient serum was pre-treated with pembro Shan Kangjia lenvatinib.

FIG. 62 shows that IL-18BP baseline serum levels may be correlated with poor response against PD-1. IL-18BP is supportive data on soluble ICP and the action of potential resistance mechanisms to PD1 blockade in melanoma cancer patients receiving anti-PD-1 treatment. High IL-18BP in serum of melanoma cancer patients pretreated with anti-PD-1 was associated with adverse responses. After gray scale normalization of the target product, raw Olink data (NPX format) student T-test was performed on IL18BP proteins.

FIGS. 63A-63B show Principal Component Analysis (PCA) of IL-18 and IL-18BP levels in serum from cervical cancer. PCA showed that mainly tumor sites were separated between samples with high and low levels of IL-18. The location of tumors in the tongue is associated with high levels of IL-18 and low levels of IL18BP, as compared to other sites.

FIG. 64 shows IL-18 and IL-18BP levels in serum from head and neck patients at different tumor sites (dot plot). Higher levels of IL-18 in serum of head and neck patients are shown in the tongue.

Figures 65A-65C show increased IL18 and IL18BP plasma levels in NSCLC patients following anti-PD-1 monotherapy or anti-PD-1+ chemotherapy combination. The mean plasma levels of IL18 and IL18BP were higher in responder patients at baseline and increased in NR patients treated with anti-PD 1. A) IL18 and IL18BP levels in plasma of R/NR NSCLC patients at baseline. B) IL18 and IL18BP levels in plasma of NSCLC patients (R/NR) alone at baseline and after single anti-PD-1 treatment. C) IL18 and IL18BP levels in plasma of NSCLC patients (R/NR) alone at baseline and after single anti-PD 1 treatment or after chemotherapy+anti-PD-1 combination treatment. D) Percentage change from IL18 and IL18BP baseline in R/NR NSCLC patients following single anti-PD-1 treatment or chemotherapy in combination with anti-PD-1. The P values in the a to C graphs were obtained after paired T-test.

Fig. 66 shows whole blood measurement data. anti-IL-18 BP antibody Ab-71709 showed no signs of systemic immune activation in id.flow (ex vivo system mimicking human blood circulation) as monotherapy or in combination with nivolumab. Fresh whole blood was taken from six healthy volunteers and immediately transferred to the whole blood circulatory system. The test item was administered and the blood was set to circulate at 37 ℃ to prevent clotting. Blood samples collected at the 24 hour time point were subjected to hematology and flow cytometer parameter analysis and then processed into plasma for cytokine analysis. The anti-CD 52 antibody alemtuzumab is included as a reference antibody due to its controlled cytokine release in the clinic. In contrast to alemtuzumab, anti-IL-18 BP antibodies did not induce any signs of systemic immune activation as monotherapy or in combination with the anti-PD 1 antibody nivolumab, depending on the various readings employed.

FIGS. 67A-67B show in vitro studies testing the effect of ADI-71739 on killing melanoma cells by human TIL. The anti-IL 18-BP antibody ADI-71739 increases the killing of melanoma cells by tumor-infiltrating lymphocytes. A) Schematic of the assay setup. MEL624 cells were co-cultured with human TIL pre-enriched with MART1 or gp100 peptide-specific clones. rhIL-18 (R & D systems,50 ng/ml) and rhIL-18BP (R & D systems, 1. Mu.g/ml) were added to the co-culture for 30 minutes to form the IL-18:IL-18BP complex prior to treatment with 10. Mu.g/ml ADI-71739 or isotype control. The co-cultures were monitored using an intucyte live cell imaging instrument for 72 hours. B) The addition of IL-18 (grey) enhanced tumor cell killing as exemplified by lower confluence (left) and increased apoptosis (right) of MEL624 cells over time. IL-18BP abrogated the effects of IL-18 in the presence of isotype control antibodies (black), while anti-IL-18 BP antibodies (blue-green) were able to fully restore these effects.

Fig. 68A-68B show in vitro studies testing the effect of ADI-71739 in combination with other checkpoint blocking antibodies. The anti-IL 18-BP antibody ADI-71739 increased CMV-specific T cell secretion IFNg both as monotherapy and in combination with aPVRIG/aTIGIT/pembrolizumab. A) Schematic of the assay setup. MEL624 cells overexpressing PD-L1 were loaded with CMV peptide pp65. Cells were incubated with rhIL-18 (R & D systems,30 ng/ml) and rhIL-18BP (R & D systems, 2. Mu.g/ml) for 30 min to form IL-18:IL-18BP complex, and then cells were treated with 10. Mu.g/ml ADI-71739 or aPVRIG (anti-PVRIG) or aTIGIT (anti-TIGIT) or pembrolizumab (anti-PD-L1) or isotype control as monotherapy or in various combinations. CMV-specific T cells were then added to the culture and IFNg secretion was measured after overnight incubation. B) anti-IL-18 BP antibodies alone were able to increase ifnγ secretion by T cells, and this effect was enhanced when combined with pembrolizumab/aPVRIG/aTIGIT.

FIGS. 69A-69B show in vitro studies testing the effect of ADI-71739 on human TIL function in the presence of endogenous IL-18BP levels. The anti-IL 18BP antibody ADI-71739 increased the release of IFNg by tumor-infiltrating lymphocytes. A) Schematic of the assay setup. MEL624 cells were co-cultured with human TIL pre-enriched with MART1 or gp100 peptide-specific clones. IL-18 (3.7 ng/ml) was added to the co-culture along with 5. Mu.g/ml ADI-71739 or isotype control. The co-culture was left for 18 hours, after which the IFNg level in the supernatant was measured. B) The level of ifnγ in co-cultures treated with ADI-71739 (agarick) was increased compared to isotype-treated samples (black). Representative examples from two TIL donors are shown.

Figures 70A-70C show that the level of bound IL-18 in TME is higher than the amount required for T cell activation in vitro. A) Schematic of the assay setup; thawed Tumor Infiltrating Lymphocytes (TILs) co-cultured with MEL624 cells at a 1:1 ratio were treated with rhIL-18 (R & D systems,1.23-300 ng/ml) for 24 hours. B) rhIL-18 in a dose dependent manner increases IFN gamma secretion. rhIL-18 activates TIL at concentrations above about 1ng/ml and reaches saturation at about 100 ng/ml. C) The levels of bound IL-18 in TDS for different indications were almost higher than required for T cell activation in vitro. The bound IL18 levels were calculated by subtracting free IL18 from total IL-18 measured for each sample by two separate ELISA kits. The red dotted line indicates the level required for functional activity (1.5 ng/gr). The black line indicates the median level of binding to IL-18 for each tumor type.

Fig. 71A-71B show that, unlike other cytokines, inflammatory body-induced cytokines such as IL-18 and IL-1B are enriched in TME. A) IL-18 and IL-1b are inflammatory small body-derived cytokines with opposite effects in TME. Although IL-18 promotes T cell and NK cell activation and leads to antitumor activity, IL1b has a dual role and leads to a pro-tumor activity in all effects. B) The dot plot shows cytokine levels in tumor derived supernatants measured in various indications. Each point represents a sample. The average is depicted by the short black line. The red dotted line indicates the limit of detection of each cytokine.

FIG. 72 shows a combination study of anti-IL-18 BP antibody and anti-PD-L1 antibody in a mouse tumor model. In a mouse tumor model, anti-IL-18 BP Ab in combination with anti-PD-L1 Ab increased tumor growth inhibition and survival. 10 6 week old female C57BL/6 mice groups were subcutaneously injected with E0771 and given a mIgG1 Synagis isotype control, anti-mouse IL-18BP Ab, anti-PDL 1 Ab or a combination of anti-mouse IL-18BP Ab and anti-PD-L1 Ab (IP), followed by 6 additional doses. Tumor volume is expressed as mean volume + SEM. Tumor volumes were measured twice weekly.

Figures 73A-73C show that administration of anti-IL 18BP is expected to have a better therapeutic window than engineering IL-18. MC38ova cells were subcutaneously injected into C57BL/6 mice and treated twice weekly with the indicated mAb SYNAGIS MIGG (IP), anti-IL 18bp mIgG1 (IP), PBS (SC) or engineered IL-18 (SC). A) Mice were weighed once a week. B) Mice were bled prior to treatment 4, 4 hours after treatment 4, and 24 hours after treatment 4. Serum was analyzed for the presence of indicator molecules-IFNg, TNF, MCP1, IL 6. C) Serum was analyzed for IL-18 levels. D. Spleens were harvested from mice 24 hours after treatment 4 and weighed. E. Spleens harvested from mice treated with either IL15 or IL15+ ILRa were weighed.

Fig. 74A-74B depict evaluation of anti-IL 18-BP monotherapy in a mouse syngeneic MC38ova tumor model. C57BL/6 mice were subcutaneously injected with 1.2M MC38ova cells and treated twice weekly with the indicated mAb SYNAGIS MIGG (IP), anti-IL 18bp mIgG1 (IP). A) Tumor growth measurements for each group, B) overview of tumor growth measurements for individual mice in each group.

FIG. 75 shows that in the MC38ova tumor model, anti-IL 18bp antibodies modulate tumor microenvironment without affecting the periphery. C57BL/6 mice were subcutaneously injected with MC38ova ^dim and treated with anti-mouse IL-18BP Ab (IP). Tumors, spleen and serum were harvested and the immune composition and cytokine concentrations were determined. A) To G) represent tumor microenvironment, H) represent spleen, I) represent serum.

MAB1191 Ab bound to human IL18BP and affinity measurements were performed using Biacore.

Fig. 77: effect of a combination of an anti-IL 18BP antibody with oxaliplatin in a MC38ovadim tumor model. 10C 57BL/6 mice were inoculated with MC38OVAdim. Mice were treated with 5mg/kg oxaliplatin or control DDW at a Tumor Volume (TV) of 110mm 3. Mice were treated with 15mg/kg of anti-IL 18BP mIgG1 Ab or isotype control followed by 5 additional doses at TV 140mm 3. (A) TV was expressed as mean volume.+ -. SEM. (B) Separate tumor measurements for each mouse are depicted (n=10 per group). CR-complete responder, PR-partial responder (TV < = 500mm 3) CR-complete responder, PR-partial responder.

FIG. 78 anti-mouse IL18BP as a single agent in MC38OVAdim and B16F10-hmgp100 mouse tumor models. 10C 57BL/6 mice were inoculated with MC38ovadim or B16F10-hmgp100 cells. Mice were treated with the indicated mabs: anti-IL-18 BP Ab or isotype control. (A-B) anti-IL-18 BP Ab inhibited tumor growth in MC38ova (A) or B16F10-hmgp (B) mouse tumor models. Tumor volumes are expressed as mean volume±sem.

Detailed Description

I. Introduction to the invention

A. interleukin 18 binding proteins

The present invention provides antibodies that specifically bind to interleukin 18 binding protein (IL 18-BP). "protein" in this context is used interchangeably with "polypeptide" and also includes peptides. The present invention provides antibodies that specifically bind to IL 18-BP.

The IL18-BP gene is located on human chromosome 11, and no exon encoding the transmembrane domain is found in the 8.3kb genomic sequence that includes the IL18-BP gene. To date, four IL18-BP isoforms have been identified in humans that result from alternative mRNA splicing. They are represented as IL18-BP a, b, C and d, which share the same N-terminus, but differ at the C-terminus (Novick, D.et al, immunity,10:127-136, (1999)). These isoforms differ in their ability to bind IL18 (Kim, S.—H. Et al, PNAS,97 (3): 1190-1195 (2000)). Among the four human IL18-BP (hIL 18-BP) isomers, isomers a and c are known to have the ability to neutralize IL18. The most enriched IL18-BP isomer (isomer a) exhibits high affinity for IL18, has a fast turn-on rate and a slow turn-off rate and a dissociation constant (K _d) of approximately 0.4nM (Kim, s.—h. Et al, PNAS,97 (3): 1190-1195 (2000)). Others have reported that IL18-BP has an affinity for IL-18 of approximately about 1pM or about 25pM (Zhou T. Et al, nature,583 (7817): 609-614, (2020), girard C. Et al, rheumatology 2016;55:2237-2247 (2016)) IL18-BPb and IL18-BPd isoforms lack the complete Ig domain and lack the ability to bind or neutralize IL18. Two murine IL18-BP isoforms produced by mRNA splicing and found in various cDNA libraries have been expressed, purified and evaluated for their ability to bind and neutralize IL18 bioactivity ((Kim, S.- -H. Et al., PNAS,97 (3): 1190-1195 (2000)). Human and mouse IL18-BP have 60.8% amino acid similarity murine IL18-BPc and IL18-BPd isoforms with the same Ig domain are also able to neutralize >95% of murine IL18. However, murine IL18-BPd with a common C-terminal motif with human IL18-BPa is also able to neutralize the large mixed electrostatic and hydrophobic binding sites in the Ig domain of human IL18-BP, which may explain their high affinity binding to ligands (Kim, S.- -H. Et al., PNAS,97 (3): 1190-1195 (2000)).

IL18-BP is a secreted protein 194 amino acids in length, with a signal peptide (spanning amino acid 1 to 30) and a secretory chain (spanning amino acid 41 to 171) and 4 potential N-glycosylation sites, but without a transmembrane domain. The full-length human IL18-BP isoform a protein is shown in FIG. 28 (SEQ ID NO: 254). The present invention provides formulations comprising antibodies that specifically bind to IL18-BP proteins. "protein" in this context is used interchangeably with "polypeptide" and also includes peptides. The present invention provides antibodies that specifically bind to IL18-BP protein. IL18-BP is a secreted protein 194 amino acids in length, with a signal peptide (spanning amino acid 1 to 30) and a secretory chain (spanning amino acid 41 to 171).

Thus, as used herein, the terms "IL18BP", "IL-18BP", "IL 18-binding protein" or "interleukin 18 binding protein" may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type IL18-BP as described herein, as well as any naturally occurring splice variant, amino acid variant or isomer. The term IL18-BP may be used interchangeably with "IL18 binding protein", "interleukin 18 binding protein", "IL18 BPa", "interleukin 18 binding protein isoform a precursor" and the term "soluble" form of IL18-BP may also be used interchangeably with the term "IL18BP soluble" or "fragment of an IL18-BP polypeptide", which may refer broadly to one or more of the following optional polypeptides. The term "soluble" with respect to the form of IL18-BP may also be used interchangeably with the term "secreted" as a fragment of an IL18-BP polypeptide, which may refer broadly to one or more of the IL18-BP polypeptides disclosed herein.

IL18-BP is constitutively expressed in the spleen and belongs to the immunoglobulin superfamily. Residues involved in the interaction of IL18 with IL18-BP were described by using computer modeling (Kim, S.—H. Et al, PNAS,97 (3): 1190-1195 (2000)) and based on the interaction between the similar proteins IL-1β and IL-1R type I (Vigers, G.P.A. et al, nature,386:190-194 (1997)). IL18-BP acts as an inhibitor of the pro-inflammatory cytokine IL 18. IL-18 regulates immune system functions including induction of IFNγ production, th1 differentiation, NK cell activation and Cytotoxic T Lymphocyte (CTL) responses (Tominga, K., et al International Immunology,12 (2): 151-160 (2000) and Senju, H., et al, int J Biol Sci.,14 (3): 331-340 (2018)). IL18-BP binds IL18, preventing binding of IL18 to its receptor, thereby inhibiting IL 18-induced T-cell and NK-cell activation and proliferation, and production of pro-inflammatory cytokines, thereby reducing T-cell and NK-cell activity and reducing T-helper type 1 immune responses. IL18-BP abrogates IL18 induction of IFN-gamma and IL18 activation of NF- κB in vitro. In addition, IL18-BP inhibits IFN-gamma induction in mice injected with LPS termini (Novick, D. Et al, immunity,10:127-136, (1999)).

IL18 is constitutively present in many cells (Puren et al, PNAS,96:2256-2261 (1999)) and circulates in healthy humans (Urushihara et al. 2000), which represents a unique phenomenon in cytokine biology. Because of the high affinity of IL18 for IL18-BP (Kd 1 pM) and the high concentration of IL18-BP found in the circulation (in excess of 20-fold molar amounts of IL 18), it is assumed that most, if not all, of the IL18 molecules in the circulation bind to IL 18-BP. Thus, circulating IL18-BP, which competes with cell surface receptors for IL18, acts as a natural anti-inflammatory and immunosuppressive molecule.

According to at least some embodiments of the invention, anti-IL 18-BP antibodies (including antigen binding fragments) that bind to IL18-BP and block the interaction of IL18 and IL18-BP, thereby releasing increased levels of free IL18, are used to enhance activation, proliferation, cytokine and/or chemokine secretion of T cells, NK cells, NKT cells, bone marrow cells, dendritic cells, MAIT cells, γδ T cells, and/or congenital lymphoid cells (ILCs) and are useful in the treatment of diseases such as cancer and pathogen infection. These anti-IL 18-BP antibodies are useful in the treatment of diseases such as cancer.

Accordingly, the present invention provides anti-IL 18-BP antibodies (e.g., including anti-IL 18-BP antibodies, including those having the same CDRs as shown in fig. 1,2, and/or 3) as provided in fig. 1,2, and/or 3. IL18-BP (also known as interleukin-18 binding protein, uniProtKB/Swiss-Prot (O95998) or HGNC (5987) NCBI Entrez Gene (10068)) involves the amino acid and nucleic acid sequences shown in RefSeq accession numbers: ng_029021.1, nm_001039659.1, np_001034748.1, nc_000011.10 chromosome reference grch38.p13 major assembly login identifier: NM_001039660.2 and NP_001034749.1 and NC_000011.9 chromosome reference GRCh38.p13 major Assembly Login identifier ：NP_001034748.1、NM_001039659.2、NP_005690.2NM_005699.3、NP_001034748.1NM_001039659.2、NP_005690.2、NM_005699.3、NP_001138529.1、NM_001145057.1、NP_001138527.1、NM_001145055.1. in some embodiments, the antibodies of the invention are specific for IL 18-BP.

Anti-IL 18-BP antibody

Accordingly, the present invention provides anti-IL 18-BP antibodies as provided in fig. 1, 2 and/or 3 (e.g., including anti-IL 18-BP antibodies, including antibodies having the same CDRs as shown in fig. 1, 2 and/or 3) and antibodies that compete for binding with the enumerated antibodies in fig. 1, 2 and/or 3.

As discussed below, the term "antibody" is generally used. Antibodies for use in the present invention may take a variety of forms as described herein, including conventional antibodies as well as antibody derivatives, fragments and mimetics as described below. Generally, the term "antibody" includes any polypeptide comprising at least one antigen binding domain, as described more fully below. As described herein, antibodies may be polyclonal, monoclonal, xenogenic, allogeneic, syngeneic, or modified versions thereof, with monoclonal antibodies having particular utility in many embodiments. In some embodiments, the antibodies of the invention specifically or substantially specifically bind to IL18-BP molecules. The terms "monoclonal antibody" and "monoclonal antibody composition" as used herein refer to a population of antibody molecules containing only one species of antigen binding sites capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody molecules containing multiple species of antigen binding sites capable of interacting with a particular antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the particular antigen with which they are immunoreactive.

Traditional full length antibody structural units typically include tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically, molecular weight of about 25 kDa) and one "heavy" chain (typically, molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention relates to the class of IgG, which has several subclasses, including but not limited to IgG1, igG2, igG3 and IgG4. Thus, as used herein, "isotype" means any subclass of immunoglobulin defined by the chemical and antigenic properties of its constant region. While exemplary antibodies denoted herein as "CPA" are based on IgG1 heavy constant regions, as shown in FIG. 4, anti-IL 18-BP antibodies of the invention include antibodies using IgG2, igG3, and IgG4 sequences, or combinations thereof. For example, as known in the art, different IgG isotypes have different effector functions, which may or may not be desirable. Thus, CPA antibodies of the invention may also replace the IgG1 constant domain with an IgG2, igG3 or IgG4 constant domain (as depicted in fig. 1E), with IgG2 and IgG4 being particularly useful in many situations, e.g., for ease of manufacture or when fewer effector functions are required, the latter being desirable in some situations.

The amino terminal portion of each chain comprises a variable region of about 100 to 110 or more amino acids (commonly referred to in the art and herein as "Fv domain" or "Fv region") that is primarily responsible for antigen recognition. In the variable region, each of the V domains of the heavy and light chains aggregates three loops to form an antigen binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a "CDR") in which the variation in amino acid sequence is most pronounced. "variable" refers to the fact that certain segments of the variable region vary widely in sequence from antibody to antibody. Variability within the variable regions is unevenly distributed. In contrast, the V region consists of a relatively constant stretch of Framework Regions (FR) called 15-30 amino acids, separated by a short region of polar variability called the "hypervariable region".

Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region typically encompasses from about amino acid residues 24 to 34 in the light chain variable region (LCDR 1;

"L" represents the light chain), amino acid residues 50 to 56 (LCDR 2) and 89 to 97 (LCDR 3) and about 31 to 35B (HCDR 1; "H" represents the heavy chain), 50 to 65 (HCDR 2) and 95 to 102 (HCDR 3) surrounding amino acid residues, but numbering is sometimes slightly offset, as will be appreciated by those skilled in the art; kabat et al SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991) and/or those residues forming hypervariable loops (e.g., residues 26 to 32 (LCDR 1), 50 to 52 (LCDR 2) and 91 to 96 (LCDR 3) in the light chain variable region and 26 to 32 (HCDR 1), 53 to 55 (HCDR 2) and 96 to 101 (HCDR 3) in the heavy chain variable region), chothia and Lesk (1987) J.mol. Biol.196:901-917. Specific CDRs of the invention are described below and shown in FIGS. 6A-6D.

The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al collected multiple primary sequences of the variable regions of the heavy and light chains. Based on the degree of conservation of the sequences, it classifies each primary sequence into CDRs and frameworks and makes a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication No. 91-3242, E.A. Kabat et al, incorporated by reference in its entirety).

In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. Herein, "immunoglobulin (Ig) domain" means an immunoglobulin region having a different tertiary structure. Of interest in the present invention are heavy chain domains, comprising a constant heavy Chain (CH) domain and a hinge domain. In the context of IgG antibodies, igG isotypes each have three CH regions. Thus, the "CH" domain in the IgG context is as follows: "CH1" means positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat.

Thus, the invention provides variable heavy domains, variable light domains, heavy constant domains, light constant domains and Fc domains as used herein as outlined. As used herein, "variable region" is meant to include one or more Ig domains that are substantially encoded by vk or vλ, and/or VH genes that constitute k, λ and heavy chain immunoglobulin genetic loci, respectively. Thus, the variable heavy domains comprise vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4 and the variable light domains comprise vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. "heavy constant region" as used herein means the CH 1-hinge-CH 2-CH3 portion of an antibody. As used herein, "Fc" or "Fc region" or "Fc domain" means a polypeptide that includes a constant region of an antibody other than the first constant region immunoglobulin domain, and in some cases includes a portion of a hinge. Thus, fc refers to the last two constant region immunoglobulin domains of IgA, igD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the N-terminal flexible hinge of these domains. For IgA and IgM, the Fc may comprise the J chain. For IgG, the Fc domain includes immunoglobulin domains cγ2 and cγ3 (cγ2 and cγ3) and a lower hinge region between cγ1 (cγ1) and cγ2 (cγ2). Although the boundaries of the Fc region may vary, a human IgG heavy chain Fc region is generally defined to comprise residues C226 or P230 at its carboxy-terminus, with numbering according to the EU index as in Kabat. In some embodiments, the Fc region is subjected to amino acid modifications, such as alterations in binding to one or more fcγr receptors or FcRn receptors, as described more fully below.

Thus, as used herein, "Fc variant" or "variant Fc" means a protein that includes amino acid modifications in the Fc domain. The Fc variants of the invention are defined in terms of the amino acid modifications constituting them. Thus, for example, N434S or 434S is an Fc variant having a serine substitution at position 434 relative to the parent Fc polypeptide, wherein numbering is according to the EU index. Similarly, M428L/N434S defines an Fc variant having substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may not be specified, in which case the variant is referred to as 428L/434S. It should be noted that the order in which the substitutions are provided is arbitrary, that is, for example, 428L/434S is the same Fc variant as M428L/N434S, or the like. For all positions discussed herein in relation to antibodies, amino acid position numbering is according to the EU index unless otherwise indicated.

"Fab" or "Fab region" as used herein means a polypeptide that includes the VH, CH1, VL and CL immunoglobulin domains. Fab may refer to this region alone or in the context of a full length antibody, antibody fragment or Fab fusion protein. As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL and VH domains of a single antibody. As will be appreciated by those skilled in the art, these are typically composed of two chains.

Throughout this specification, reference is made to residues in the variable domain (generally, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), typically using either the IMTG numbering system or the Kabat numbering system (e.g., kabat et al, supra (1991)). EU numbering as in Kabat is typically used for constant domains and/or Fc domains.

CDRs facilitate antigen binding or more specifically epitope binding site formation of antibodies. An "epitope" refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. An epitope is a group of molecules (grouping) such as amino acids or sugar side chains and generally has specific structural properties as well as specific charge properties. A single antigen may have more than one epitope.

Epitopes can include amino acid residues that are directly involved in binding (also referred to as immunodominant components of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen binding peptide; in other words, the amino acid residues are within the footprint of the peptide to which the antigen is specifically bound.

Epitopes may be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is a linear epitope produced by adjacent amino acid residues in a polypeptide chain. Conformational epitopes may differ from non-conformational epitopes in that binding to the former but not to the latter is lost in the presence of denaturing solvents.

Epitopes typically comprise at least 3 and more commonly at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies recognizing the same epitope can be validated with a simple immunoassay that shows the ability of one antibody to block binding of another antibody to a target antigen, e.g. "binning". Specific binning is described below.

The definition of "antibody" includes the "antigen-binding portion" of an antibody (also used interchangeably with "antigen-binding fragment", "antibody fragment" and "antibody derivative"). That is, for the purposes of the present invention, the antibodies of the present invention have minimal functional requirements for binding to the IL18-BP antigen. As understood by those of skill in the art, there are a number of antigen fragments and derivatives that retain the ability to bind antigen, but still have alternative structures, including but not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) F (ab') 2 fragments, including bivalent fragments of two linked Fab fragments, (vii) single chain Fv molecules (scFv), wherein VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al, 1988,Science 242:423-426, huston et al, 1988, proc. Natl. Acad. Sci. U.S. 85:5879-5883, all by incorporation), (iv) "diabodies" or "triabodies" (iii) multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al, 2000,Methods Enzymol.326:461-479; WO 94/04), holliger et al, 1993, acad. Sci. Sci.Sci.Sci.5, sci.Sci.5, or "bodies" (and other species such as monoclonal antibodies from the family of the group consisting of monoclonal antibodies, the single-dAbs.48, and the whole camelid bodies "(e.g., monoclonal antibodies, scdAbs.48, scFv, and other antibodies).

Still further, the antibody or antigen binding portion thereof (antigen binding fragment, antibody portion) may be part of a larger immunoadhesion molecule (sometimes also referred to as a "fusion protein") formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of immunoadhesion molecules include the use of the streptavidin core region to make tetrameric scFv molecules, and the use of cysteine residues, tag peptides and C-terminal polyhistidine tags to make bivalent and biotinylated scFv molecules. Antibody portions, such as Fab and F (ab') ₂ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein.

Typically, the anti-IL 18-BP antibodies of the invention are recombinants. As used herein, "recombinant" broadly refers to a product (e.g., a cell, nucleic acid, protein, or vector) that has been modified by the introduction of a heterologous nucleic acid or protein or alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

As used herein, the term "recombinant antibody" encompasses all antibodies that are produced, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) Antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) an antibody isolated from a recombinant combinatorial human antibody library; and (d) antibodies produced, expressed, produced or isolated by any other means that involves splicing the human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may undergo in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the V _H region and V _L region of the recombinant antibodies are the following sequences: although derived from and related to human germline V _H sequences and V _L sequences, may not naturally occur in the human antibody germline repertoire in vivo.

A. anti-IL 18-BP binding antibodies

The invention provides anti-IL 18-BP antibodies. (for convenience, "anti-IL 18-BP antibody" and "IL18-BP antibody" are used interchangeably). The anti-IL 18-BP antibodies of the invention specifically bind to human IL18-BP, and preferably to the secretory chain of human IL18-BP, as depicted in FIG. 28, including, for example, anti-IL 18-BP antibodies, including antibodies having the same CDRs as shown in FIGS. 1, 2 and 3.

As described herein and more fully below, anti-IL 18-BP antibodies (including antigen binding fragments) that simultaneously bind to IL18-BP and block the interaction of IL18-BP and IL18, thereby releasing increased levels of free IL18, are used to enhance activation, proliferation, cytokine and/or chemokine secretion of T cells, NK cells, NKT cells, bone marrow cells, dendritic cells, MAIT T cells, γδ T cells, and/or congenital lymphoid cells (ILCs) and are useful in the treatment of diseases such as cancer and pathogen infection.

Specific binding to IL18-BP or an IL18-BP epitope may be expressed, for example, by an antibody having a KD of: at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M, at least about 10 ^-13 M, at least about 10 ^-14 M, or greater, wherein KD refers to the rate of dissociation of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, 100,000-fold, or more than a control molecule relative to an IL18-BP antigen or epitope.

However, as supported by the examples, for optimal binding to IL18-BP, antibodies preferably have KD (also referred to as binding affinity) of less than 0.01nM, less than 10nM, and most preferably less than 0.1pM, less than 1pM, less than 0.1pM, and less than 0.01pM, can be used in the methods of the invention. In some embodiments, the anti-IL-18 BP exhibits a KD of less than 900pM, less than 850pM, less than 800pM, less than 750pM, less than 700pM, less than 650pM, less than 600pM, less than 550pM, less than 500pM, less than 450pM, less than 400pM, less than 350pM, less than 300pM, less than 250pM, less than 200pM, less than 150pM, less than 100pM, less than 50pM, or less than 10 pM. In some embodiments, the anti-IL-18 BP antibody exhibits a KD of less than 750 pM. In some embodiments, an anti-IL 18-BP antibody of the invention binds to human IL18-BP with 50nM or less, 10nM or less, or 1nM or less (i.e., higher binding affinity), 100pM or less, 10pM or less, 1pM or less, 0.1pM or less, or 0.01pM or less, K _D, wherein K _D is determined by known methods (e.g., surface plasmon resonance (SPR, e.g., biacore instrument), ELISA, kinExA, and most typically SPR at 25 ℃ or 37 ℃). In some embodiments, the anti-IL 18-BP antibodies of the invention bind to human IL18-BP with a KD of: less than 900pM, less than 850pM, less than 800pM, less than 750pM, less than 700pM, less than 650pM, less than 600pM, less than 550pM, less than 500pM, less than 450pM, less than 400pM, less than 350pM, less than 300pM, less than 250pM, less than 200pM, less than 150pM, less than 100pM, less than 50pM, or less than 10pM, wherein K _D is determined by known methods (e.g., surface plasmon resonance (SPR, e.g., biacore instrument), ELISA, kinex a, and most typically SPR at 25 ℃ or 37 ℃). In some embodiments, the antibody preferably has the following KD or binding affinity: less than 0.005pM、0.01pM、0.02pM、0.03pM、0.04pM、0.05pM、0.06pM、0.07pM、0.08pM、0.09pM、0.10pM、0.15pM、0.20pM、0.25pM、0.30pM、0.35pM、0.40pM、0.45pM、0.50pM、0.55pM、0.60pM、0.65pM、0.70pM、0.75pM、0.80pM、0.85pM、0.90pM、0.95pM or 1pM.

Furthermore, specific binding of a particular antigen or epitope may be manifested, for example, by an antibody directed against KA or KA of the IL18-BP antigen or epitope being: at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, 100,000-fold, or more than the epitope relative to the control, wherein KA or KA refers to the association rate of a particular antibody-antigen interaction.

The present invention provides antigen binding domains, including full length antibodies, comprising a number of specific, enumerated 6 CDR sets, as provided in fig. 1, 2 and/or 3. The present invention provides antigen binding domains, including full length antibodies, comprising several specific, enumerated 6 CDR sets, as provided in fig. 3.

The invention also provides variable heavy and light chain domains and full length heavy and light chains.

As discussed herein, the invention also provides variants of the above components, including variants in CDRs, as described above. In addition, when Fc variants are used, the variable heavy chain may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a "VH" sequence herein, and/or comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more amino acid changes. Variable light chains are provided that may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a "VL" sequence herein, and/or comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more amino acid changes when Fc variants are used. Similarly, heavy and light chains are provided that may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the "HC" and "LC" sequences herein, and/or comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more amino acid changes when Fc variants are used.

Thus, the present invention provides antibodies, typically full length or scFv domains, comprising the following CHA sets of CDRs, the sequences of which are shown in fig. 1-3.

CDRs were generated using ADI-71701, ADI-71709, ADI-71710, ADI-71707 and ADI-71717 antibodies to the 66650 lineage (VH 1-03; VL-kappa-1-5) consensus sequence (FIG. 1A). The corresponding sequence alignment is shown in FIG. 3B.

The 66650 lineage (VH 1-03; VL-kappa-1-5) consensus sequences include:

● CDR-H1 having the sequence Y-T-F-X-X2-Y-A-X3-H, wherein X is N, R, D, G or K; x2 is S, H, I or Q; x3 is M or V;

● CDR-H2 having the sequence W-I-H-A-G-T-G-X-T-X2-Y-S-Q-K-F-Q-G, wherein X is N, A or V; x2 is K or L;

● CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is S or E;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence E-A-S-S-L-E-S; and

● CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is S, V, Y, L or Q; x2 is F, S or G.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence E-A-S-S-L-E-S; and

CDRs were generated using ADI-71719, ADI-71720, ADI-71722 and ADI-71728 antibodies for the 66670 lineage (VH 1-69; VL-kappa-1-12) consensus sequences (FIG. 1B). The corresponding sequence alignment is shown in FIG. 3C.

The 66670 lineage (VH 1-69; VL-kappa-1-12) consensus sequences include:

● CDR-H1 having the sequence G-T-F-X-X2-Y-X3-I-S, wherein X is S or N; x2 is E or S; x3 is V or P;

● CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is G or Y and X2 is A or S; x3 is N, I or V;

● CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S;

● CDR-L3 having the sequence Q-Q-V-Y-X-X2-P-W-T, wherein X is S or R; x2 is L, I or F.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

The 66692 lineage (VH 3-23, VL-kappa-1-12) consensus sequences for CDRs were generated using ADI-71662, ADI-71663 and ADI-66692 antibodies (FIG. 1C). The corresponding sequence alignment is shown in FIG. 3A.

The 66692 lineage (VH 3-23, VL-kappa-1-12) consensus sequences include:

● CDR-H1 having the sequence F-T-F-X-N-X2-A-M-S, wherein X is G or D or S; x2 is T or V or Y;

● CDR-H2 having the sequence A-I-S-X-X1-X2-G-S-T-Y-Y-A-D-S-V-K-G, wherein X is G or A; x2 is N or S; x3 is A or G;

● CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

● CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

● CDR-L3 having the sequence Q-H-A-X-X1-F-P-Y-T, wherein X is Y or L; x2 is S or F.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

● CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

The 66716 lineage (VH 1-39; VL-kappa-1-12) consensus sequences for CDRs were generated using ADI-71736, ADI-71739 and ADI-66716 antibodies (FIG. 1D). The corresponding sequence alignment is shown in fig. 3D.

The 66716 lineage (VH 1-39; VL-kappa-1-12) consensus sequences include:

● CDR-H1 having the sequence G-S-I-S-S-X-X2-Y-X3-W-G, wherein X is S or P; x2 is E or D; x3 is G, P or Y;

● CDR-H2 having the sequence S-I-X-X2-X3-G-X4-T-Y-Y-N-P-S-L-K-S, wherein X is Y or V; x2 is Y or N; x3 is Q or S; x4 is S or A;

● CDR-H3 having the sequence A-R-G-P-X-R-Q-X2-F-D-Y, wherein X is Y or H and X2 is V or L;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

● CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is S or F; x2 is S or V.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H1 having the sequence Y-T-F-X-X2-Y-A-X3-H, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

● CDR-H2 having the sequence W-I-H-A-G-T-G-X-T-X2-Y-S-Q-K-F-Q-G, wherein X is any amino acid; x2 is any amino acid;

● CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is any amino acid;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence E-A-S-S-L-E-S; and

● CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H1 having the sequence G-T-F-X-X2-Y-X3-I-S, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

● CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is any amino acid and X2 is any amino acid;

● CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is any amino acid; ● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S;

● CDR-L3 having the sequence Q-Q-V-Y-X-X2-P-W-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

● CDR-L2 having the sequence A-A-S-S-L-Q-S;

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H1 having the sequence F-T-F-X-N-X2-A-M-S, wherein X is any amino acid; x2 is any amino acid;

● CDR-H2 having the sequence A-I-S-X-X1-X2-G-S-T-Y-Y-A-D-S-V-K-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

● CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

● CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is any amino acid

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

● CDR-L3 having the sequence Q-H-A-X-X1-F-P-Y-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, an anti-IL 18-BP antibody includes CDRs:

● CDR-H1 having the sequence G-S-I-S-S-X-X2-Y-X3-W-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid;

● CDR-H2 having the sequence S-I-X-X2-X3-G-X4-T-Y-Y-N-P-S-L-K-S, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid; x4 is any amino acid;

● CDR-H3 having the sequence A-R-G-P-X-R-Q-X2-F-D-Y, wherein X is any amino acid; x2 is any amino acid;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

● CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is any amino acid; x2 is any amino acid.

In some embodiments, the antibody comprises:

C) CDR-H3 having the sequence A-R-G-L-G-X-V-G-P-T-G-T-S-W-F-D-P, wherein X is S, L, A, K or E; a light chain variable domain, the light chain variable domain comprises:

d) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

E) CDR-L2 having the sequence E-A-S-S-E-S, wherein X is L or S; and

F) CDR-L3 having the sequence Q-Q-Y-R-X-X2-P-F-T, wherein X is S, V, Y, L, T or Q; x2 is F, S, Y or G.

In some embodiments, the antibody comprises:

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F; and

A light chain variable domain, the light chain variable domain comprises:

d) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

E) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

F) CDR-L3 having the sequence Q-Q-X-Y-X2-X3-P-W-T, wherein X is V or L; x2 is S or R; x3 is L, I or F.

In some embodiments, the antibody comprises:

d) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

E) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

F) CDR-L3 having the sequence Q-H-X-X2-X3-F-P-Y-T, wherein X is A or G; x2 is Y, R or L; x3 is S, R, L or F.

In some embodiments, the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

d) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

D) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

E) CDR-L3 having the sequence Q-Q-G-X-X2-F-P-Y-T, wherein X is S, N, W or F; x2 is S or V.

Anti-IL 18-BP antibodies also include a framework region. The framework regions of the variable heavy and variable light chains may be humanized as known in the art (occasional variants in CDRs, if desired) so that humanized variants of the VH and VL chains of fig. 1,2 and/or 3 may be produced. In addition, the humanized variable heavy and light chain domains can then be fused to human constant regions, such as those from IgG1, igG2, igG3, and IgG 4.

In addition, sequences that may have identical CDRs but vary in the variable domain (or the entire heavy or light chain) are also included. For example, IL18-BP antibodies include antibodies having the same CDRs as shown in fig. 1-3, but may be less identical along the variable region, e.g., 85%, 88%, 90%, 92%, 95%, or 98% identical. For example, IL18-BP antibodies include antibodies having the same CDRs as shown in fig. 3, but may be less identical along the variable region, e.g., 95% or 98% identical and in some embodiments at least 95% or at least 98%.

The percentage identity between two amino acid sequences can be calculated using the following algorithm that has been incorporated into the ALIGN program (version 2.0): e.meyers and w.miller (comput. Appl. Biosci.,4:11-17 (1988)) were determined using PAM120 weight residue table, gap length penalty 12, and gap penalty 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (j. Mol. Biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program in the GCG software package (commercially available) using the Blossum 62 matrix or PAM250 matrix and GAP weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1,2,3, 4, 5 or 6.

Additionally or alternatively, the protein sequences of the invention may also be used as "query sequences" to search public databases, for example, to identify related sequences. Such retrieval may be performed using the following XBLAST program (version 2.0): altschul et al (1990) J.mol.biol.215:403-10. According to at least some embodiments of the invention, BLAST protein searches may be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to antibody molecules of the invention. To obtain a gapped alignment for comparison purposes, gapped BLAST may be utilized, as described in the following: altschul et al, (1997) Nucleic Acids Res.25 (17): 3389-3402. When utilizing BLAST programs and gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Typically, the percent identity between IL18-BP antibodies for comparison is at least 75%, at least 80%, at least 90%, preferably at least about 95%, 96%, 97%, 98% or 99% percent identity. The percent identity may be along the entire amino acid sequence, e.g., the entire heavy or light chain or along a portion of the chain. For example, included within the definition of anti-IL 18-BP antibodies of the invention are those antibodies that share identity along the entire variable region (e.g., wherein the identity along the variable region is 95% or 98%, and in some embodiments at least 95% or at least 98%), or along the entire constant region, or along the Fc domain alone.

B. Specific anti-IL 18-BP antibodies

The present invention provides antigen binding domains, including full length antibodies, comprising several specific, enumerated sets of 6 CDRs, as well as shared CDRs (see, e.g., those listed in fig. 1A-1D).

The antibodies described herein are labeled as follows. Antibodies have reference numbers such as "66650 lineage (VH 1-03; VL-kappa-1-5)" or "VH1-03" or "ADI-71663hIgG 4S 228 Pkappa". This represents a CDR and/or a combination of variable heavy and variable light chains, as depicted in fig. 1,2 and/or 3. "ADI-71663.VH" refers to the variable heavy portion of ADI-71663hIgG 4S 228Pκ, while "ADI-71663.VL" is the variable light chain. "ADI-71663.vhCDR1", "ADI-71663.vhCDR2", "ADI-71663.vhCDR3", "ADI-71663.vlCDR1", "ADI-71663.vlCDR2" and "ADI-71663.vlCDR3" refer to the indicated CDRs. "ADI-71663.HC" refers to the entire heavy chain (e.g., variable domain and constant domain) of the molecule, and "ADI-71663.LC" refers to the entire light chain (e.g., variable domain and constant domain) of the same molecule.

In many embodiments, the antibodies of the invention are human (derived from phage) and block IL18-BP. As shown in fig. 1A to 1D and fig. 2A to 2P, the anti-IL 18-BP antibodies and their components are also summarized below:

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 from the 66650 lineage (VH 1-03; VL-kappa-1-5);

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 from the 66670 lineage (VH 1-69; VL-kappa-1-12);

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 from the 66692 lineage (VH 3-23, VL-kappa-1-12);

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 from the 66716 lineage (VH 1-39; VL-kappa-1-12);

ADI-71663, ADI-71663.VH, ADI-71663.VL, ADI-71663.HC, ADI-71663.LC and ADI-71663.H1、ADI-71663.H2、ADI-71663.H3、ADI-71663.H4;ADI-71663.vhCDR1、ADI-71663.vhCDR2、ADI-71663.vhCDR3、ADI-71663.vlCDR1、ADI-71663.vlCDR2 and ADI-71663.VlCDR3;

ADI-71662, ADI-71662.VH, ADI-71662.VL, ADI-71662.HC, ADI-71662.LC and ADI-71662.H1、ADI-71662.H2、ADI-71662.H3、ADI-71662.H4;ADI-71662.vhCDR1、ADI-71664.71662、ADI-71662.vhCDR3、ADI-71662.vlCDR1、ADI-71662.vlCDR2 and ADI-71662.vlCDR3;

ADI-71701, ADI-71701.VH, ADI-71701.VL, ADI-71701.HC, ADI-71701.LC and ADI-71701.H1、ADI-71701.H2、ADI-71701.H3、ADI-71701.H4;ADI-71701.vhCDR1、ADI-71701.vhCDR2、ADI-71701.vhCDR3、ADI-71701.vlCDR1、ADI-71701.vlCDR2 and ADI-71701.VlCDR3;

ADI-71709, ADI-71709.VH, ADI-71709.VL, ADI-71709.HC, ADI-71709.LC and ADI-71709.H1、ADI-71709.H2、ADI-71709.H3、ADI-71709.H4;ADI-71709.vhCDR1、ADI-71709.vhCDR2、ADI-71709.vhCDR3、ADI-71709.vlCDR1、ADI-71709.vlCDR2 and ADI-71709.vlCDR3;

ADI-71710, ADI-71710.VH, ADI-71710.VL, ADI-71710.HC, ADI-71710.LC and ADI-71710.H1、ADI-71710.H2、ADI-71710.H3、ADI-71710.H4;ADI-71710.vhCDR1、ADI-71710.vhCDR2、ADI-71710.vhCDR3、ADI-71710.vlCDR1、ADI-71710.vlCDR2 and ADI-71710.VlCDR3;

ADI-71719, ADI-71719.VH, ADI-71719.VL, ADI-71719.HC, ADI-71719.LC and ADI-71719.H1、ADI-71719.H2、ADI-71719.H3、ADI-71719.H4;ADI-71719.vhCDR1、ADI-71719.vhCDR2、ADI-71719.vhCDR3、ADI-71719.vlCDR1、ADI-71719.vlCDR2 and ADI-71719.VlCDR3;

ADI-71720, ADI-71720.VH, ADI-71720.VL, ADI-71720.HC, ADI-71720.LC and ADI-71720.H1、ADI-71720.H2、ADI-71720.H3、ADI-71720.H4;ADI-71720.vhCDR1、ADI-71720.vhCDR2、ADI-71720.vhCDR3、ADI-71720.vlCDR1、ADI-71720.vlCDR2 and ADI-71720.VlCDR3;

ADI-71722, ADI-71722.VH, ADI-71722.VL, ADI-71722.HC, ADI-71722.LC and ADI-71722.H1、ADI71722.H2、ADI-71722.H3、ADI-71722.H4、ADI-71722.vhCDR1、ADI-71722.vhCDR2、ADI-71722.vhCDR3、ADI-71722.vlCDR1、ADI-71722.vlCDR2 and ADI-71722.VlCDR3;

ADI-71717, ADI-71717.VH, ADI-71717.VL, ADI-71717.HC, ADI-71717.LC and ADI-71717.H1、ADI71717.H2、ADI-71717.H3、ADI-71717.H4;ADI-71717.vhCDR1、ADI-71717.vhCDR2、ADI-71717.vhCDR3、ADI-71717.vlCDR1、ADI-71717.vlCDR2 and ADI-71717.vlCDR3;

ADI-71739, ADI-71739.VH, ADI-71739.VL, ADI-71739.HC, ADI-71739.LC and ADI-71739.H1、ADI71739.H2、ADI-71739.H3、ADI-71739.H4;ADI-71739.vhCDR1、ADI-71739.vhCDR2、ADI-71739.vhCDR3、ADI-71739.vlCDR1、ADI-71739.vlCDR2 and ADI-71739.VlCDR3;

ADI-71736, ADI-71736.VH, ADI-71736.VL, ADI-71736.HC, ADI-71736.LC and ADI-71736.H1、ADI71736.H2、ADI-71736.H3、ADI-71736.H4;ADI-71736.vhCDR1、ADI-71736.vhCDR2、ADI-71736.vhCDR3、ADI-71736.vlCDR1、ADI-71736.vlCDR2 and ADI-71736.vlCDR3;

ADI-71707, ADI-71707.VH, ADI-71707.VL, ADI-71707.HC, ADI-71707.LC and ADI-71707.H1、ADI-71707.H2、ADI-71707.H3、ADI-71707.H4;ADI-71707.vhCDR1、ADI-71707.vhCDR2、ADI-71707.vhCDR3、ADI-71707.vlCDR1、ADI-71707.vlCDR2 and ADI-71707.VlCDR3;

AB-837, AB-837.VH, AB-837.VL, AB-837.HC, AB-837.LC and AB-837.H1、AB-837.H2、AB-837.H3、AB-837.H4;AB-837.vhCDR1、AB-837.vhCDR2、AB-837.vhCDR3、AB-837.vlCDR1、AB-837.vlCDR2 and AB-837.VlCDR3;

ADI-66692, ADI-66692.VH, ADI-66692.VL, ADI-66692.HC, ADI-66692.LC and ADI-66692.H1、ADI-66692.H2、ADI-66692.H3、ADI-66692.H4;ADI-66692.vhCDR1、ADI-66692.vhCDR2、ADI-66692.vhCDR3、ADI-66692.vlCDR1、ADI-66692.vlCDR2 and ADI-66692.vlCDR3;

ADI-66716, ADI-66716.VH, ADI-66716.VL, ADI-66716.HC, ADI-66716.LC and ADI-66716.H1、ADI-66716.H2、ADI-66716.H3、ADI-66716.H4;ADI-66716.vhCDR1、ADI-66716.vhCDR2、ADI-66716.vhCDR3、ADI-66716.vlCDR1、ADI-66716.vlCDR2 and ADI-66716.VlCDR3;

ADI-71728, ADI-71728.VH, ADI-71728.VL, ADI-71728.HC, ADI-71728.LC and ADI-71728.H1、ADI-71728.H2、ADI-71728.H3、ADI-71728.H4;ADI-71728.vhCDR1、ADI-71728.vhCDR2、ADI-71728.vhCDR3、ADI-71728.vlCDR1、ADI-71728.vlCDR2 and ADI-71728.VlCDR3; or alternatively

ADI-71741, ADI-71741.VH, ADI-71741.VL, ADI-71741.HC, ADI-71741.LC and ADI-71741.H1、ADI71741.H2、ADI-71741.H3、ADI-71741.H4;ADI-71741.vhCDR1、ADI-71741.vhCDR2、ADI-71741.vhCDR3、ADI-71741.vlCDR1、ADI-71741.vlCDR2 and ADI-71741.vlCDR3;

ADI-71742, ADI-71742.VH, ADI-71742.VL, ADI-71742.HC, ADI-71742.LC and ADI-71742.H1、ADI71742.H2、ADI-71742.H3、ADI-71742.H4;ADI-71742.vhCDR1、ADI-71742.vhCDR2、ADI-71742.vhCDR3、ADI-71742.vlCDR1、ADI-71742.vlCDR2 and ADI-71742.VlCDR3;

ADI-71744, ADI-71744.VH, ADI-71744.VL, ADI-71744.HC, ADI-71744.LC and ADI-71744.H1、ADI71744.H2、ADI-71744.H3、ADI-71744.H4;ADI-71744.vhCDR1、ADI-71744.vhCDR2、ADI-71744.vhCDR3、ADI-71744.vlCDR1、ADI-71744.vlCDR2 and ADI-71744.vlCDR3;

ADI-71753, ADI-71753.VH, ADI-71753.VL, ADI-71753.HC, ADI-71753.LC and ADI-71753.H1、ADI71753.H2、ADI-71753.H3、ADI-71753.H4;ADI-71753.vhCDR1、ADI-71753.vhCDR2、ADI-71753.vhCDR3、ADI-71753.vlCDR1、ADI-71753.vlCDR2 and ADI-71753.VlCDR3; or alternatively

ADI71755, ADI-71755.VH, ADI-71755.VL, ADI-71755.HC, ADI-71755.LC and ADI-71755.H1、ADI71755.H2、ADI-71755.H3、ADI-71755.H4;ADI-71755.vhCDR1、ADI-71755.vhCDR2、ADI-71755.vhCDR3、ADI-71755.vlCDR1、ADI-71755.vlCDR2 and ADI-71755.VlCDR3.

A. IL18-BP antibodies that compete for binding with enumerated antibodies

The present invention provides not only enumerated antibodies, but also additional antibodies that compete with the enumerated antibodies (VH and ADI numbering enumerated herein that specifically bind IL 18-BP) for specific binding to IL18-BP molecules. IL18-BP antibodies of the invention include antibodies that bind to one or more of the enumerated antibodies, including VH1-03.66650、VH1-69.66670、VH3-23.66692、VH1-39.66716、VL-κ-1-5-66650、VL-κ-1-12、66670、VL-κ-1-12、66692、VL-κ-1-12、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755.

B. Production of additional antibodies

Additional antibodies to human IL18-BP may be raised using well-known methods, such as those outlined in the examples, as is well known in the art. Thus, additional anti-IL 18-BP antibodies can be generated by conventional methods, such as immunization of mice (sometimes using DNA immunization, e.g., such as used by Aldevron), followed by screening for the production of anti-IL 18-BP (including human IL 18-BP) proteins and hybridomas, and antibody purification and recovery.

C. optional antibody engineering

The anti-IL 18-BP antibodies of the invention (e.g., anti-IL 18-BP antibodies, including antibodies having the same CDRs as shown in fig. 1,2, and/or 3) can be modified or engineered to alter the amino acid sequence by amino acid substitutions.

"Amino acid substitution" or "substitution" herein means the replacement of an amino acid at a particular position in the parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur at a particular location or within an organism or any organism. For example, substitution E272Y refers to a variant polypeptide, in this case an Fc variant, wherein the glutamic acid at position 272 is replaced with tyrosine. For clarity, proteins that have been engineered to alter the nucleic acid coding sequence but not the starting amino acid (e.g., CGG (encoding arginine) to CGA (still encoding arginine) to increase the expression level of the host organism) are not "amino acid substitutions"; that is, although a novel gene encoding the same protein is produced, if the protein has the same amino acid at a specific position at which it starts, the protein is not an amino acid substitution.

Amino acid substitutions may be made to alter the affinity of the CDRs for the IL18-BP protein (including both increasing and decreasing binding, as more fully outlined below), as well as to alter additional functional properties of the antibody, as discussed herein. For example, antibodies can be engineered to include modifications within the Fc region, typically used to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, antibodies according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation to once again alter one or more functional properties of the antibody. Such embodiments are further described below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In some embodiments, the hinge region of C _H1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain junction region of the Fc-hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In some embodiments, amino acid substitutions may be made in the Fc region, typically in order to alter binding to fcγr receptors. As used herein, "fcγreceptor," "fcγr," "or fcγr" means any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by fcγr genes. In humans, this family includes, but is not limited to, fcyri (CD 64), including the isoforms fcyria, fcyrib, and fcyric; fcγrii (CD 32), comprising isoforms fcγriia (comprising isoforms H131 and R131), fcγriib (comprising fcγriib-1 and fcγriib-2), and fcγriic; and FcgammaRIII (CD 16), comprising the isomers FcgammaRIIIa (comprising isoforms V158 and F158) and FcgammaRIIIb (comprising isoforms FcgammaRIIIb-NA 1 and FcgammaRIIIb-NA 2) (Jefferis et al, 2002, immunology report (Immunol Lett) 82:57-65, incorporated by reference in its entirety), as well as any undiscovered human FcgammaR or FcgammaR isomers or isoforms. Fcγr can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. Mouse fcγrs include, but are not limited to, fcγri (CD 64), fcγrii (CD 32), fcγriii-1 (CD 16) and fcγriii-2 (CD 16-2), and any undiscovered mouse fcγr or fcγr isomer or allotype.

There are a number of useful Fc substitutions that can be made to alter binding to one or more of the fcγr receptors. Substitutions that result in increased binding as well as decreased binding may be useful. For example, it is known that increasing binding to fcγriiia generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; in which non-specific cytotoxic cells expressing fcγr recognize antibodies bound on target cells and subsequently cause cell-mediated reactions of lysis of the target cells, similarly, in some cases, amino acid substituents useful in the present invention include those listed in U.S. Ser. No. 11/124,620 (particularly FIG. 41) and U.S. Pat. No. 6,737,056, which are expressly incorporated herein by reference in their entirety and particularly the variants disclosed therein.

In addition, the antibodies of the invention are modified to increase their biological half-life. Various methods are possible. For example, one or more of the following mutations may be introduced: T252L, T254,254, 254S, T256F as described in U.S. patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody may be altered within the C _H1 or C _L region to contain a salvage receptor binding epitope taken from both loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. nos. 5,869,046 and 6,121,022 to presa et al. Additional mutations for increasing serum half-life are disclosed in U.S. Pat. nos. 8,883,973, 6,737,056 and 7,371,826 and include 428L, 434A, 434S and 428L/434S.

In yet other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be substituted with different amino acid residues such that the antibody has an altered affinity for the effector ligand but retains the antigen binding capacity of the parent antibody. The affinity altered effector ligand may be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both of which are Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be substituted with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC). This method is described in further detail in U.S. Pat. No. 6,194,551 to Idusogie et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This method is further described in PCT publication WO 94/29351 to Bodmer et al.

In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity ：238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438 or 439 of the antibody for fcγ receptors by modifying one or more amino acids at the following positions. This method is further described in PCT publication WO 00/42072 to Presta. Furthermore, binding sites for FcgammaRI, fcgammaRII, fcgammaRIII and FcRn have been mapped on human IgG1 and variants with improved binding have been described (see Shields, R.L. et al (2001) J.biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to FcyRIII. In addition, the following combination mutants are shown to increase fcyriii binding: T256A/S298A, S A/E333A, S A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and increase antibody circulating half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

In yet another embodiment, the antibody may be modified to eliminate in vivo Fab arm exchange. In particular, the process involves the exchange of IgG4 half-molecules (one heavy chain plus one light chain) between other IgG4 antibodies, effectively producing functionally monovalent bispecific antibodies. Mutations in the heavy chain hinge region and constant domain can eliminate this exchange (see Aalberse, RC, schuurman j.,2002,Immunology 105:9-19).

In yet another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies can be prepared (i.e., antibodies lack glycosylation). Glycosylation can be altered, for example, to increase the affinity of an antibody for an "antigen" or to reduce effector functions such as ADCC. Such carbohydrate modification may be accomplished, for example, by altering one or more glycosylation sites (e.g., N297) within the antibody sequence. For example, one or more amino acid substitutions may be made which result in the elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.

Additionally or alternatively, antibodies with altered glycosylation patterns, such as low fucosylation antibodies with reduced fucose residues or antibodies with increased bisecting GlcNac structure, can be prepared. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modification may be accomplished, for example, by expressing the antibody in a host cell having an altered glycosylation mechanism. Cells having altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies according to at least some embodiments of the invention are expressed to thereby produce antibodies having altered glycosylation. For example, cell lines Ms704, ms705 and Ms709 lack the fucosyltransferase gene FUT8 (α (1, 6) -fucosyltransferase), such that antibodies expressed in the Ms704, ms705 and Ms709 cell lines lack fucose on their carbohydrates. Ms704, ms705 and Ms709 FUT8 cell lines were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two alternative vectors (see Yamane et al and U.S. patent publication No. 20040110704 (2004) Biotechnol Bioeng 87:87:614-22) to Yamane-Ohnuki et al. As another example, EP 1,176,195 of Hanai et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such cell line exhibit low fucosylation by reducing or eliminating alpha 1,6 linkage-associated enzymes. Hanai et al also describe cell lines with low enzymatic activity for the addition of fucose to N-acetylglucosamine, with or without enzymatic activity, in combination with the Fc region of antibodies, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT publication WO 03/035835 to Presta describes a variant CHO cell line, i.e., lec13 cells, which has a reduced capacity to link fucose to Asn (297) linked carbohydrates, which also causes hypofucosylation of antibodies expressed in the host cells (see also Shields, R.L. et al (2002) J.biol. Chem. 277:26733-26740). PCT publication WO 99/54342 to Umana et al describes cell lines engineered to express glycoprotein modified glycosyltransferases (e.g., beta (1, 4) -N-acetylglucosamine transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structure, which increases ADCC activity of the antibodies (see also Umana et al (1999) Nat. Biotech.17:176-180). Alternatively, fucosidase may be used to cleave fucose residues of antibodies. For example, fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A.L. et al (1975) biochem. 14:5516-23).

Another modification of the antibodies herein contemplated by the present invention is pegylation or the addition of other water-soluble moieties, typically polymers, for example to increase half-life. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. For pegylation of antibodies, the antibodies or fragments thereof are typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derive other proteins, such as mono (C ₁-C₁₀) alkoxy-polyethylene glycol or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a deglycosylated antibody. Methods for pegylating proteins are known in the art and may be applied to antibodies according to at least some embodiments of the invention. See, for example, EP 0 154 316 to Nishimura et al and EP 0 401 384 to Ishikawa et al.

In addition to substitutions to alter binding affinity to fcγrs and/or FcRn and/or increase serum half-life in vivo, additional antibody modifications may be made, as described in further detail below.

In some cases, affinity maturation is performed. Amino acid modifications in CDRs are sometimes referred to as "affinity maturation". An "affinity matured" antibody is an antibody having one or more alterations in one or more CDRs which results in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not have those one or more alterations. In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is generally not preferred.

In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of an IL18-BP antibody of the invention. Typically, only 1,2 or 3 amino acids are substituted in any single CDR and no changes of more than 1,2,3, 4, 5, 6, 7, 8, 9 or 10 are typically made within a set of CDRs. However, it should be understood that any combination of unsubstituted, 1,2, or 3 substitutions in any CDR may be independently and optionally combined with any other substitution.

Affinity maturation may be performed such that the binding affinity of the antibody to the IL18-BP antigen is increased by at least about 100% or more, or at least about 10 ⁴ or more, 10 ⁵ or more, 10 ⁶ or more, 10 ⁷ or more, as compared to the "parent" antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the IL18-BP antigen. Affinity matured antibodies were generated by known procedures. See, e.g., marks et al 1992,Biotechnology 10:779-783, which describe affinity maturation by shuffling of Variable Heavy (VH) and Variable Light (VL) domains. Random mutagenesis of CDRs and/or framework residues is described, for example, in: barbas, et al, PNAS, USA 91:3809-3813 (1994); shier et al, gene,169:147-155 (1995); yelton et al, J.Immunol.,155:1994-2004 (1995); jackson et al, J.Immunol.154 (7): 3310-9 (1995); and Hawkins et al, J.mol.biol.,226:889-896 (1992).

Alternatively, "silent" amino acid modifications may be made in one or more CDRs of an antibody of the invention, e.g., without significantly altering the affinity of the antibody for an antigen. The reasons for this are numerous, including optimized expression (as may be done with nucleic acids encoding antibodies of the invention).

Thus, included within the definition of CDRs and antibodies of the invention are variant CDRs and antibodies; that is, an antibody of the invention may comprise amino acid modifications in one or more of the CDRs of an enumerated antibody of the invention. In addition, amino acid modifications may also be made independently and optionally in any region outside the CDRs (including framework regions and constant regions), as outlined below.

Nucleic acid compositions

Nucleic acid compositions encoding the anti-IL 18-BP antibodies of the invention are also provided, as are expression vectors containing the nucleic acids and host cells transformed with the nucleic acids and/or the expression vector compositions. As will be appreciated by those of skill in the art, due to the degeneracy of the genetic code, the protein sequences depicted herein may be encoded by any number of possible nucleic acid sequences.

The nucleic acid composition encoding the IL18-BP antibody will depend on the format of the antibody. Traditionally, tetrameric antibodies comprising two heavy chains and two light chains are encoded by two different nucleic acids, one nucleic acid encoding the heavy chain and one nucleic acid encoding the light chain. As known in the art, they may be placed into a single expression vector or two expression vectors, and transformed into a host cell where they are expressed to form an antibody of the invention. In some embodiments, for example when using scFv constructs, a single nucleic acid encoding a variable heavy chain-linker-variable light chain is typically used, which can be inserted into an expression vector for transformation into a host cell. The nucleic acid may be placed into an expression vector containing appropriate transcriptional and translational control sequences including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, and the like.

Preferred mammalian host cells for expressing recombinant antibodies according to at least some embodiments of the invention include chinese hamster ovary (CHO cells), per.c6, HEK293 and other cells known in the art.

The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkali/SDS treatment, csCl strips, column chromatography, agarose gel electrophoresis, and other techniques well known in the art.

To generate scFv genes, the DNA fragment encoding V _H and V _L is operably linked to another fragment encoding a flexible linker, e.g., encoding an amino acid sequence (Gly ₄-Ser)₃ (SEQ ID NO: 150), such that the V _H and V _L sequences may be expressed as a continuous single chain protein, with the V _L region and V _H region being linked by a flexible linker (see, e.g., bird et al, science 242:423-426 (1988); huston et al PNAS,85:5879-5883 (1988); mcCafferty et al Nature348:552-554 (1990)).

Administration of anti-IL 18-BP antibody formulations

Administration of pharmaceutical compositions comprising an anti-IL 18-BP antibody of the invention (e.g., an anti-IL 18-BP antibody, including antibodies having the same CDRs as shown in fig. 1, 2, and/or 3), preferably in the form of a sterile aqueous solution, can be performed in a variety of ways, as is known in the art, protein therapeutics are typically delivered by IV infusion. Antibodies of the invention may also be delivered using such methods. For example, administration may be intravenous injection or intravenous infusion with 0.9% sodium chloride as an infusion carrier. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit 1980.

In some embodiments, the dosage and frequency of administration is selected to be therapeutically or prophylactically effective. As known in the art, it may be necessary to adjust protein degradation, systemic and local delivery, new protease synthesis rate, and age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and such adjustment will be determinable by one of skill in the art through routine experimentation. For the treatment of a patient, a therapeutically effective dose of an Fc variant of the invention may be administered. By "therapeutically effective dose" herein is meant a dose administered to produce an effect.

V. administration of anti-IL 18-BP antibody formulations

Administration of pharmaceutical compositions comprising an anti-IL 18-BP antibody of the invention (e.g., an anti-IL 18-BP antibody, including antibodies having the same characteristics as those shown in fig. 1,2, and/or 3), preferably in the form of a sterile aqueous solution, can be performed in a variety of ways, as known in the art, with protein therapeutics typically delivered by IV infusion. Antibodies of the invention may also be delivered using such methods. For example, administration may be intravenous injection or intravenous infusion with 0.9% sodium chloride as an infusion carrier. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit 1980.

Methods of using anti-IL 18-BP antibodies

A. therapeutic agent use

Anti-IL 18-BP antibodies (e.g., anti-IL 18-BP antibodies, including the antibodies described in fig. 1,2, and/or 3) may be used to treat patients, such as human subjects, that typically have a disorder associated with IL18-BP or free IL18 levels. As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, as a matter of example, involving the treatment of cancer. Those in need of treatment include those already with cancer, as well as those in which cancer is to be prevented. Thus, a mammal to be treated herein may have been diagnosed with cancer, and may also be predisposed to having or susceptible to cancer. As used herein, the term "treatment" refers to preventing, delaying onset, curing, reversing, alleviating, minimizing, inhibiting, stopping the deleterious effects of, or stabilizing the discernible symptoms of the cancerous diseases, disorders, or conditions described above. It also includes the management of cancer as described above. By "managing" is meant reducing the severity of a disease, reducing the frequency of attacks of a disease, reducing the duration of such attacks, reducing the severity of such attacks, slowing/reducing the growth or proliferation of cancer cells, slowing the progression of at least one symptom, improving at least one measurable physical parameter, and the like. For example, an immunostimulatory anti-IL 18-BP immune molecule should promote immunization of T cells, NK cells, NKT cells, bone marrow cells, dendritic cells, MAIT cells, γδ T cells and/or congenital lymphoid cells (ILCs) or cytokines directed against target cells (e.g., cancer, infected or pathogen cells) to treat cancer or infectious diseases by depleting cells involved in the disease state.

The IL18-BP antibodies of the invention are provided in therapeutically effective doses. A "therapeutically effective dose" of an anti-IL 18-BP immune molecule according to at least some embodiments of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease-free symptomatic periods, an increase in longevity, disease remission, or prevention or reduction of injury or disability due to affliction with a disease. For example, for treatment of an IL18-BP positive tumor, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to an untreated subject. The ability of a compound to inhibit tumor growth can be assessed in an animal model system that predicts the efficacy of a human tumor. Alternatively, such properties of the composition may be assessed by examining the ability of the compound to inhibit, such in vitro inhibition by assay is known to the skilled practitioner. A therapeutically effective amount of the therapeutic compound may reduce tumor size or otherwise alleviate symptoms in the subject.

One of ordinary skill in the art will be able to determine a therapeutically effective amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

1. Cancer treatment

The IL18-BP antibodies of the invention have particular use in the treatment of cancer, alone or in combination with other therapeutic agents. In general, the antibodies of the invention are immunomodulatory in that the anti-IL 18-BP antibodies of the invention typically stimulate the immune system by inhibiting the action of IL18-BP, rather than directly attacking cancer cells. Thus, unlike tumor-targeted therapies which aim to inhibit molecular pathways critical to tumor growth and development and/or deplete tumor cells, cancer immunotherapy aims to stimulate the patient's own immune system to deplete cancer cells, thereby providing long-term tumor destruction. Various methods are available for cancer immunotherapy, including therapeutic cancer vaccines that induce tumor-specific T cell responses, and immunostimulatory antibodies that eliminate immunosuppressive pathways (i.e., antagonists of inhibitory receptors = immune checkpoints).

The clinical response of targeted therapies or conventional anti-cancer therapies is often transient, as cancer cells develop resistance and tumor recurrence occurs. However, in the past few years, the clinical application of cancer immunotherapy has shown that this type of treatment can have a durable clinical response, which has shown a significant impact on long-term survival. However, while the response is long-term, only a few patients respond (in contrast to conventional or targeted therapies, in which a large number of patients respond, but the response is transient).

When a tumor is clinically detected, it has escaped the immune defense system by acquiring immunological resistance and immunosuppressive properties and producing an immunosuppressive tumor microenvironment through various mechanisms and various immune cells.

Thus, the anti-IL 18-BP antibodies of the invention are useful for the treatment of cancer. Due to the nature of the mechanism of action of immune tumors, IL18-BP does not necessarily need to be over-expressed or associated with a particular cancer type; that is, the objective is to have anti-IL 18-BP antibodies remove inhibition of activation of T cells, NK cells, NKT cells, bone marrow cells, dendritic cells, MAIT T cells, γδ T cells, and/or congenital lymphoid cells (ILCs), thereby allowing the immune system to track cancer.

As used herein, "cancer" refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division (e.g., uncontrolled cell growth) that causes malignant growth or a tumor. As used herein, the term "cancer" or "cancerous" is to be understood to include any neoplastic disease (whether invasive, non-invasive, or metastatic) characterized by abnormal and uncontrolled cell division that causes malignant growth or a tumor, non-limiting examples of which are described herein. This includes any physiological condition in mammals that is normally characterized by unregulated cell growth.

"Cancer therapy" as used herein refers to any method of preventing or treating cancer or ameliorating one or more of the symptoms of cancer. Typically, such therapies will include administering an immunostimulatory anti-IL 18-BP antibody (including antigen binding fragments) alone or in combination with chemotherapy or radiation therapy or other biological agents to enhance its activity, i.e., activity in individuals in which expression of IL18-BP inhibits an anti-tumor response and the efficacy or biological efficacy of the chemotherapy or radiation therapy.

As part of the monotherapy or combination therapy described herein, the anti-IL 18-BP antibodies of the invention may be used to treat solid tumors (including, for example, lung cancer, liver cancer, breast cancer, brain cancer, gastrointestinal cancer) and blood cancers (including, for example, leukemia and pre-leukemia diseases, lymphomas, plasma cell diseases), carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. In some embodiments, the cancer is early. In some embodiments, the cancer is advanced (including metastatic). In some embodiments, cancers suitable for treatment of the invention include cancers that express IL18-BP, and also include non-metastatic or non-invasive as well as invasive or metastatic cancers, including cancers in which expression of IL18-BP by immune, mesenchymal or diseased cells inhibits both an anti-tumor response and an anti-invasive immune response. In some embodiments, anti-IL 18-BP antibodies may be used to treat vascularized tumors. In some embodiments, cancers treated with the anti-IL 18-BP antibodies of the invention include carcinomas, lymphomas, sarcomas, and/or leukemias. In some embodiments, cancers for treatment with the anti-IL 18-BP antibodies of the invention include vascularized tumors, melanomas, non-melanoma skin cancers (squamous cell carcinoma and basal cell carcinoma), mesothelioma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine lung cancer (including pleural mesothelioma, neuroendocrine lung cancer), NSCL (large cell), NSCLC large cell adenocarcinoma, non-small cell lung cancer (NSCLC), NSCLC squamous cell carcinoma, soft tissue sarcoma, kaposi's sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, NSCLC with PDL1> = 50% TPS, Neuroendocrine lung cancer, atypical carcinoid lung cancer, peritoneal cancer, esophageal cancer, hepatocellular cancer, liver cancer (including HCC), gastric cancer (GASTRIC CANCER), gastric cancer (stomach cancer) (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, urothelial cancer, bladder cancer, liver cancer, glioma, brain cancer (and oedema, such as oedema associated with brain tumor), breast cancer (including, for example, triple negative breast cancer), testicular cancer, testicular germ cell tumor, colon cancer, colorectal cancer (CRC), colorectal cancer MSS (MSS-CRC); Refractory MSS colorectal cancer; MSS (microsatellite steady state), primary peritoneal carcinoma, primary peritoneal ovarian carcinoma, microsatellite stabilized primary peritoneal carcinoma, platinum-resistant microsatellite stabilized primary peritoneal carcinoma, CRC (MSS unknown), rectal cancer, endometrial carcinoma (endometrial cancer) (including endometrial carcinoma (endometrial carcinoma)), uterine carcinoma, salivary gland carcinoma, renal cell carcinoma (RENAL CELL CANCER, RCC), renal cell carcinoma (RENAL CELL carcinoma, RCC), renal cell carcinoma (endometrial cancer), Gastroesophageal junction cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, carcinoid, head and neck cancer, B-cell lymphomas including non-Hodgkin's lymphoma, low grade/follicular non-Hodgkin's lymphoma, small Lymphocyte (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, diffuse large B-cell lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-nucleated cells NHL, giant tumor NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemiaMacroglobulinemia), hodgkin's lymphoma (HD), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute Myelogenous Leukemia (AML), hairy cell leukemia, chronic myeloblastic leukemia, multiple myeloma, post-transplant lymphoproliferative disease (PTLD), abnormal vascular proliferation associated with mole-type hamartoma, meigs' syndrome, merkel cell carcinoma, high MSI carcinoma, KRAS mutant tumors, adult T-cell leukemia/lymphoma, adenoid cystic carcinoma (including adenoid cystic carcinoma), melanoma, malignant melanoma, metastatic melanoma, pancreatic carcinoma, ovarian carcinoma (ovarian cancer) (including ovarian carcinoma (ovarian carcinoma)), pleural mesothelioma, cervical squamous cell carcinoma (neck SCC), anal SCC), non-primary carcinoma, uterine carcinoma, pleural mesothelioma, endometrial carcinoma, chondrosarcoma, endometrial sarcoma, astrocytoma, fibroid carcinoma, and Anaplastic (AL), and Amyloidosis (AL).

In some embodiments, the cancer for treatment with an anti-IL 18-BP antibody of the invention comprises a cancer selected from the group consisting of: clear cell carcinoma (RCC), lung cancer, NSCLC, lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple Negative Breast Cancer (TNBC), head and neck tumors, colorectal adenocarcinoma, melanoma, and metastatic melanoma.

2. Anti-IL-18 BP antibody monotherapy

The IL-18BP antibodies of the invention find particular use in the treatment of cancer as monotherapy. Due to the nature of the mechanism of action of immune tumors, IL18-BP does not necessarily need to be over-expressed or associated with a particular cancer type; that is, the goal is to have anti-IL 18-BP antibodies remove inhibition of T cell and NK cell activation, thereby allowing the immune system to track cancer.

Any of the anti-IL-18 antibodies of fig. 1-3 may be used as a monotherapy.

3. Anti-IL 18BP antibody combination therapies

As known in the art, combination therapies comprising therapeutic antibodies targeting immunotherapeutic targets and additional therapeutic agents against disease conditions hold great promise. For example, in the field of immunotherapy, there are several promising combination therapies using chemotherapeutic agents (small molecule drugs or anti-tumor antibodies) or immune tumor antibodies.

The terms "in combination with … …" and "co-administration" are not limited to administration of the prophylactic or therapeutic agent at exactly the same time. Rather, it means that the antibody and the other agent or agents are administered in a sequence and at intervals such that they can act together to provide increased benefits over treatment with the antibody or other agent or agents of the invention alone. Preferably, the effects of the antibody and the other agent or agents are additive, and particularly preferably they act synergistically.

Thus, the antibodies of the invention may be administered concurrently with one or more other therapeutic regimens or agents. In some embodiments, the antibodies of the invention are administered in the same formulation as one or more other therapeutic regimens or agents. In some embodiments, the antibodies of the invention are administered in separate and/or distinct formulations from one or more other therapeutic regimens or agents. Additional therapeutic regimens or agents may be used to increase the efficacy or safety of the antibody. Moreover, additional treatment regimens or agents may be used to treat the same disease or co-condition, rather than altering the effect of the antibody. For example, an antibody of the invention may be administered to a patient in conjunction with chemotherapy, radiation therapy, or both.

In some embodiments, the anti-IL 18 BP antibodies of the invention may be combined with one of several checkpoint receptor antibodies. In some embodiments, receptor expression of a tumor in a patient may be assessed and the results then used to inform a clinician which antibody to administer. Any of the anti-IL-18 antibodies of fig. 1-3 may be used as part of a combination therapy.

A. Immune checkpoint inhibitor combination therapy

In some embodiments, the combination or composition further comprises an additional active agent, e.g., a second antigen binding protein. Optionally, the second antigen binding protein binds to a negative regulator, immunosuppressant, or immune checkpoint protein of the immune system, including but not limited to PD-1, PD-L1, CTLA-4, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, PVR, LAG, CD112, PVRIG, CD96, TIM3, and/or BTLA, or a co-stimulatory receptor: ICOS, OX40, 41BB, CD27, and/or GITR. All patent documents listed in the following sections are incorporated herein by reference in their entirety for all purposes.

In some embodiments, an anti-IL 18-BP antibody is used in combination with an antibody to an immune checkpoint inhibitory protein. In some embodiments, the immune checkpoint inhibitory protein is selected from the group consisting of: anti-PVRIG antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-CTLA-4 antibodies, anti-PD-L2 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-CEACAM-1 antibodies, anti-PVR antibodies, anti-LAG 3 antibodies, anti-CD 112 antibodies, anti-CD 96 antibodies, anti-TIM 3 antibodies, anti-BTLA antibodies, anti-ICOS antibodies, anti-OX 40 antibodies or anti-41 BB antibodies, anti-CD 27 antibodies or anti-GITR antibodies.

In some embodiments, anti-IL 18-BP antibodies are used in combination with one or more anti-PD-1 antibodies (e.g., anti-PD-1 targeting antibodies), including, for example, but not limited to, nivolumab @BMS; CHECKMATE 078) and pembrolizumabMerck), TSR-042 (Tesaro), cimipn Li Shan anti (REGN 2810; regeneron Pharmaceuticals, see US 20170174779), BMS-936559, swadazumab (PDR 001, novartis), dermatitid (CT-011; pfizer Inc.), tirelimumab (BGB-A317, beiGene), carilimumab (SHR-1210, incyte and Jiangsu HengRui), SHR-1210 (CTR 20170299 and CTR 20170322), SHR-1210 (CTR 20160175 and CTR 20170090), xindi Li Shan antibodyElily and Innovent Biologics), terlipressin Li Shan (JS 001, shanghai Junshi Bioscience), JS-001 (CTR 20160274), IBI308 (CTR 20160735), BGB-A317 (CTR 20160872), pari An Puli mab (AK 105, akeso Biopharma), sapalimab (Arcus), BAT1306 (Bio-Thera Solutions Ltd), Sago Li Shan anti (PF-06809591, pfizer), duotalizumab (Dostarlimab-gxly) (GlaxoSmithKline LLC), palo Li Shan anti (Biocad), caldonil Li Shan anti (Akeso Inc), jilo Li Shan anti (Genor BioPharma Co Ltd), st Lu Lishan anti (Shanghai Henlius Biotech Inc), baterimumab (Agenus Inc), raffin Li Shan anti (Incyte Corp), raffin, Celizumab (Johnson & Johnson), CS-1003 (EQRx Inc), IBI-318 (Innovent Biologics Inc), ivortizumab (Akeso Inc), pratelizumab (Lepu Biopharma Co Ltd), QL-1604 (Qilu Pharmaceutical Co Ltd), SCTI-10A (SinoCelltech Group Ltd), terpolizumab (MacroGenics Inc), and, AZD-7789 (AstraZeneca Plc), bragg Li Shan anti (AbbVie Inc), EMB-02 (EpimAb Biotherapeutics Inc), ebenicillin (Boehringer Ingelheim International GmbH)、F-520(Shandong New Time Pharmaceutical Co Ltd)、HX-009(Waterstone Hanxbio Pty Ltd)、Zeluvalimab(Amgen)、Peresolimab(Eli Lilly and Co)、 Luo Sini Li Shan anti (AnaptysBio Inc), ewy Su Shan anti (Xencor), izuralimab (Xencor), loregex Li Shan anti (MacroGenics Inc), and, YBL-006 (Y-Biologics Inc) as well as ONO-4635 (Ono Pharmaceutical Co Ltd), LY-3434172 (ELI LILLY AND Co) and/or PD-1 antibodies as set forth in US2017/0081409 and other antibodies being developed, which may be used in combination with the anti-IL 18BP antibodies of the invention. Additional exemplary anti-PD-1 antibody sequences are shown in fig. 39.

In some embodiments, pembrolizumab is administered at a dose of about 2mg/kg to 10 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 2 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 2 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 3 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 4 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 5 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 6 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 7 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 8 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 9 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 10 mg/kg.

In some embodiments, pembrolizumab is administered at a dose of about not more than 2 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 1mg/kg to 2 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 0.1mg/kg to 1 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 0.01mg/kg to 0.1 mg/kg.

In some embodiments, pembrolizumab is administered at a dose of about at least 10 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 10mg/kg to 20 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 20mg/kg to 30 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 30mg/kg to 40 mg/kg. In some embodiments, pembrolizumab is administered at a dose of about 40mg/kg to 50 mg/kg.

In some embodiments, pembrolizumab is administered about once every 1 week to every 6 weeks. In some embodiments, pembrolizumab is administered about once per week. In some embodiments, pembrolizumab is administered about once every 2 weeks. In some embodiments, pembrolizumab is administered about once every 3 weeks. In some embodiments, pembrolizumab is administered about once every 4 weeks. In some embodiments, pembrolizumab is administered about once every 5 weeks. In some embodiments, pembrolizumab is administered about once every 6 weeks.

In some embodiments, pembrolizumab is administered at a dose of about 2mg/kg once every 3 weeks. In some embodiments, pembrolizumab is administered at a dose of about 10mg/kg once every 3 weeks. In some embodiments, pembrolizumab is administered at a dose of about 200mg once every 3 weeks. In some embodiments, pembrolizumab is administered at a dose of about 400mg once every 6 weeks.

In some embodiments, pembrolizumab is administered within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 25 minutes, within about 30 minutes, within about 35 minutes, or within about 40 minutes. In some embodiments, pembrolizumab is administered within about 30 minutes +/-10 minutes.

In addition, the disclosure of pembrolizumab is provided below ：https://www.accessdata.fda.gov/spl/data/157262d6-15e0-4b0a-968f-b99bab4aef50/157262d6-15e0-4b0a-968f-b99bab4aef50.xml,, the entire contents of which are incorporated herein by reference.

In some embodiments, an anti-IL 18-BP antibody is used in combination with one or more anti-PD-L1 antibodies (e.g., anti-PD-L1 targeting antibodies). There are three approved anti-PD-L1 antibodies, alemtuzumab @MPDL3280A; IMpower110,110; roche/Genentech, avermectinMSB001071 8C; EMD serrono & Pfizer) and dewaruzumab (MEDI 4736; AstraZeneca). And other antibodies being developed, such as lodalimab (LY 3300054, eli Lily), pi Weishan anti (Jounce Therapeutics Inc), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), en Wo Lishan anti (Jiangsu Simcere Pharmaceutical Co Ltd), shu Geli mab (CStone Pharmaceuticals Co Ltd), ke Xili mab (Checkpoint Therapeutics Inc), parker Mi Lishan anti (CytomX Therapeutics Inc)、IBI-318、IBI-322、IBI-323(Innovent Biologics Inc)、INBRX-105(Inhibrx Inc)、KN-046(Alphamab Oncology)、6MW-3211(Mabwell Shanghai Bioscience Co Ltd)、BNT-311(BioNTech SE)、FS-118(F-star Therapeutics Inc)、GNC-038(Systimmune Inc)、GR-1405(Genrix(Shanghai)Biopharmaceutical Co Ltd)、HS-636(Zhejiang Hisun Pharmaceutical Co Ltd)、LP-002(Lepu Biopharma Co Ltd)、PM-1003(Biotheus Inc)、PM-8001(Biotheus Inc)、STIA-1015(ImmuneOncia Therapeutics LLC)、ATG-101(Antengene Corp Ltd)、BJ-005(BJ Bioscience Inc)、CDX-527(Celldex Therapeutics Inc)、GNC-035(Systimmune Inc)、GNC-039(Systimmune Inc)、HLX-20(Shanghai Henlius Biotech Inc)、JS-003(Shanghai Junshi Bioscience Co Ltd)、LY-3434172(Eli Lilly and Co)、MCLA-145(Merus NV)、MSB-2311(Transcenta Holding Ltd)、PF-07257876(Pfizer Inc)、Q-1802(QureBio Ltd)、QL-301(QLSF Biotherapeutics Inc)、QLF-31907(Qilu Pharmaceutical Co Ltd)、RC-98(RemeGen Co Ltd)、TST-005(Transcenta Holding Ltd)、 atuzumab (IMpower 133), BMS-936559/MDX-1105 and/or RG-7446/MPDL3280A and/or yw243.55.s70. In some embodiments, the PD-L1 antibody is one described in U.S. patent publication No. 2017/0281764 and WO 2013/079174 (avermectin) and WO 2010/077634 (or US2016/0222117 or US 8,217,149; alemtuzumab). In some embodiments, PD-L1 antibodies include the heavy chain sequence of SEQ ID NO:34 and the light chain sequence of SEQ ID NO:36 (from US 2017/281764), as well as other sequences being developed, which may be used in combination with the anti-IL 18BP antibodies of the invention. Additional exemplary anti-PD-L1 antibody sequences are shown in figure 40.

In some embodiments, an anti-IL 18-BP antibody is used in combination with one or more anti-PD-L2 antibodies (e.g., anti-PD-L2 targeting antibodies). Examples of anti-PD-L2 antibodies include, for example, but are not limited to, anti-PD-L2 antibodies as described in WO 2010/036959, anti-PD-L2 antibodies as described in WO 20140/22758, and other antibodies being developed, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-CTLA-4 antibodies (e.g., anti-CTLA-4 targeting antibodies). Examples of anti-CTLA-4 antibodies include, for example, but are not limited to, the FDA approved antibodies ipilimumab or tremelimumab. In some embodiments, anti-CTLA-4 antibodies include, for example, but are not limited to(Ipilimumab or antibody 10D1, described in PCT publication WO 01/14424), tremelimumab (original name tiximab, CP-675,206), monoclonal or anti-CTLA-4 antibodies described by any of the following publications: WO 98/42752; WO 00/37504; U.S. patent No. 6,207,156; hurwitz et al (1998) Pro.Natl.Acad.Sci.USA 95 (17): 10067-10071; camacho et al (2004) J.Clin.Oncology 22 (145): antibodiestract No.2505 (antibody CP-675206); and Mokyr et al (1998) Cancer Res.58:5301-5304. Any anti-CTLA-4 antibody disclosed in WO 2013/173223, as well as other antibodies being developed, may also be used, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-B7H 3 antibodies (e.g., anti-B7H 3 targeting antibodies). Examples of anti-B7H 3 antibodies include antibodies in clinical studies, such as enoxaab (MGA 271; macroGenics) and anti-B7H 3 antibodies as described in WO 2016/033225, anti-B7H 3 antibodies as outlined in US 9,441,049, and other antibodies under development, which may be used in combination with the anti-IL 18BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-B7H 4 antibodies (e.g., anti-B7H 4 targeting antibodies). Examples of anti-B7H 4 antibodies include, for example, but are not limited to, anti-B7H 4 monoclonal antibodies from FIVEPRIME, FPA150, which is currently in clinical phase I, antibodies as described in WO 2022/002012, and other antibodies being developed, which may be used in combination with the anti-IL 18BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-carcinoembryonic antigen-related cell adhesion molecule-1 antibodies (also known as anti-CEACAM 1 antibodies or anti-CD 66a antibodies). Examples of anti-CEACAM-1 antibodies include, for example, but are not limited to, antibodies in clinical studies, such as Bei Suoshan antibodies (THERAPHARM), AMG211 (Amgen), and CM-24 (MK-6018, kitovpharma). Examples of anti-CEACAM-1 antibodies also include antibodies as outlined in US20200277398A1 (CM-24 under development by FAMEWAVE LTD), antibodies as outlined in US 9072797B2 (CD 66 binding component and radionuclide yttrium-90 (90Y)), and other antibodies under development, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-PVR antibodies (e.g., anti-PVR targeting antibodies). Examples of anti-PVR antibodies include, for example and without limitation, antibodies as described in WO 2017/149538, anti-PVR antibodies include antibodies as described in WO 2021/070181. In some embodiments, the second agent is selected from one or more of the antagonists of PVRL1, PVRL2, PVRL3, PVRL4, and CD155, such as ASG-22CE (ASTELLAS PHARM/a Inc), enfrazumab (ASTELLAS PHARMA), and other drugs being developed, which can be used in combination with an anti-IL 18-BP antibody of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-LAG 3 antibodies (e.g., anti-LAG 3 targeting antibodies). Examples of anti-LAG 3 antibodies include, for example, but are not limited to, antibodies in clinical studies, e.g., LAG525 (Novartis), TSR-033 (Tesaro), furin mab (REGN 3767, regeneron), BI-754111 (Boehringer Ingelheim), sym-022 (Symphogen), RO7247669 (Roch), BMS-986016 (see WO 2010/019570), GSK2831781 (see US 2016/0017037) and Merck clone 22D2, 11C9, 4a10 and/or 19E8 (see WO 2016/028672), and antibodies comprising CDRs or the variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which antibodies are described in US 2011/0150892, WO 2010/19570 and WO 2014/008218. Other art-recognized anti-LAG-3 antibodies that may be used include IMP731 and IMP-321 described in US 2011/007033, WO 2008/132601, and WO 2009/44273. anti-LAG-3 antibodies that compete with and/or bind to the same epitope as any of these antibodies, as well as other antibodies being developed that can be used in combination with the anti-IL 18-BP antibodies of the invention, can also be used in combination therapies.

In some embodiments, anti-IL 18-BP antibodies are used in combination with one or more anti-CD 112 (also known as PVRL2; and include, for example, anti-CD 112 targeting antibodies)) antibodies. Examples of anti-CD 112 antibodies include, for example, but are not limited to, anti-CD 112 antibodies as outlined in US2020/0040081, anti-CD 112 antibodies as outlined in US2019/0040154 or anti-CD 112 antibodies as outlined in WO 2017/021526, as well as other antibodies being developed, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-CD 96 antibodies (e.g., anti-CD 96 targeting antibodies). Examples of anti-CD 96 antibodies include, for example, but are not limited to, anti-CD 96 antibodies as outlined in WO 2019/091449, anti-CD 96 antibodies as outlined in WO 2021042019, and other antibodies under development, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

For some embodiments, an anti-IL 18-BP antibody is used in combination with one or more anti-TIM 3 antibodies (e.g., anti-TIM 3 targeting antibodies). Examples of anti-TIM 3 antibodies include antibodies in clinical studies, such as Sabatolimab(Novartis)、TSR-022(Tesaro)、INCAGN02385(Incyte Corporation)、INCAGN02390(Incyte Corporation)、BGB-A425(BeiGene)、LY3321367(Eli Lilly)、BMS986258, and other antibodies under development, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-BTLA antibodies (e.g., anti-BTLA targeting antibodies). Examples of anti-BTLA antibodies include, for example, but are not limited to, JS004 (Shanghai Junshi Bioscience), anti-BTLA antibodies disclosed in WO 2011/014438, and other antibodies being developed, which can be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-ICOS antibodies (e.g., anti-ICOS targeting antibodies). Examples of anti-ICOS antibodies include, for example, but are not limited to, anti-ICOS antibodies in clinical studies, such as MEDI-570 (MedImmune), voparimab (Jounce Therapeutics), KY1044 (Kymab Limited), feixosmithkline. Examples of anti-ICOS antibodies also include anti-ICOS antibodies as outlined in US 9,957,323, anti-ICOS antibodies as outlined in WO 2016/120789, anti-ICOS antibodies as outlined in WO 2016/154177, and other antibodies being developed, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-OX 40 antibodies (e.g., anti-OX 40 targeting antibodies). Examples of anti-OX 40 antibodies include, for example, but are not limited to, anti-OX 40 antibodies in clinical studies, examples of anti-OX 40 antibodies such as PF-04518600(Pfizer)、BAT6026(Bio-Thera Solutions)、MEDI6469、MEDI-0562、MEDI6962(MedImmune)、BMS 986178、GSK3174998、ABBV-368(AbbVie)、ATOR-1015(Alligator Bioscience). also include anti-OX 40 antibodies as outlined in US10,730,951, anti-OX 40 antibodies as outlined in US10,851,173, and other antibodies being developed, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-41 BB antibodies (e.g., anti-41 BB targeting antibodies). Examples of anti-41 BB antibodies include, for example, but are not limited to Wu Tuolu mab (Pfizer, PF-05082566), LVGN6051 (Lyvgen Biopharma), ATOR-1017 (Alligator Bioscience), BMS-663513, anti-41 BB antibodies as outlined in US10,501,551, and other antibodies being developed, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-CD 27 antibodies (e.g., anti-CD 27 targeting antibodies). Examples of anti-CD 27 antibodies include, for example, but are not limited to, valirudin (CDX-1127,Leap Therapeutics), anti-CD 27 antibodies as outlined in US2020/0277393, anti-CD 27 antibodies as outlined in WO 2019/195452, and other antibodies under development, which may be used in combination with the anti-IL 18-BP antibodies of the invention.

In some embodiments, an anti-IL 18-BP antibody is used in combination with one or more anti-GITR antibodies (e.g., anti-GITR targeting antibodies). Examples of clinical studies of anti-GITR antibodies include, for example, but are not limited to, MK-4166、MK-1248(Merck Sharp&Dohme)、BMS-986156、INCAGN01876(Incyte Corporation)、OMP-336B11(OncoMed Pharmaceuticals)、MEDI1873(MedImmune). examples of anti-GITR antibodies further include, but are not limited to, anti-GITR antibodies as described in WO 2016/196792, anti-GITR antibodies as described in WO 2015/187835 (the contents of which are incorporated herein by reference), e.g., antibodies having the heavy and light chain variable regions, CDRs, heavy and light chain variable regions, or heavy and light chains of antibodies 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and/or 6G10, and variants thereof. The antibody sequences described in WO 2015/187835 are provided in Table 2 (see SEQ ID NOS: 5-14 and 27-228). The patient may also be treated with any other anti-GITR antibody, such as ,RX518(Leap Therapeutics)、MK-4166(Merck)、LKZ-145(Novartis)、GWN-323(Novartis Pharmaceuticals Corp.)、Medi1873(Medlmmune)、INBRX-110(Inhibrx)、GITR-Fc proteins (OncoMed) and WO 2006/105021、WO 2009/009116、WO 2011/028683、US2014/0072565、US2014/0072566、US2014/0065152、WO 2015/031667、WO 2015/184099、WO 2015/184099 or the antibodies described in WO 2016/054638.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-TIGIT antibodies. Examples of anti-TIGIT antibodies include, for example, but are not limited to CPA.9.083.H4(S241P)、CPA.9.086.H4(S241P)、CHA.9.547.7.H4(S241P)、CHA.9.547.13.H4(S241P)、CPA.9.018、CPA.9.027、CPA.9.049、CPA.9.057、CPA.9.059、CPA.9.083、CPA.9.086、CPA.9.089、CPA.9.093、CPA.9.101、CPA.9.103、CHA.9.536.3.1、CHA.9.536.3、CHA.9.536.4、CHA.9.536.5、CHA.9.536.7、CHA.9.536.8、CHA.9.560.1、CHA.9.560.3、CHA.9.560.4、CHA.9.560.5、CHA.9.560.6、CHA.9.560.7、CHA.9.560.8、CHA.9.546.1、CHA.9.547.1、CHA.9.547.2、CHA.9.547.3、CHA.9.547.4、CHA.9.547.6、CHA.9.547.7、CHA.9.547.8、CHA.9.547.9、CHA.9.547.13、CHA.9.541.1、CHA.9.541.3、CHA.9.541.4、CHA.9.541.5、CHA.9.541.6、CHA.9.541.7 and cha.9.541.8, cha.9.547.18 as disclosed in WO 2018/220446, and other antibodies in clinical studies, such as EOS-448 (GlaxoSmithKline, iTeos Therapeutics), BMS-986207, multi-valvulizumab (AB 154, arcus Biosciences, inc.) AB308 (Arcus Bioscience), european-Fabry-Li Shan antibody (aBGB-A1217, beiGene), tiarey Li Youshan antibody (MTIG7192A,Roche)、BAT6021(Bio-Thera Solutions)、BAT6005(Bio-Thera Solutions)、IBI939(Innovent Biologics,US2021/00040201)、JS006(Junshi Bioscience/COHERUS)、ASP8374(Astellas Pharma Inc)、 vitamin Bo Li Shan antibody (MK-7684,Merck Sharp&Dohme), M6332 (MERCK KGAA), ai Tili mab (OMP-313M32,Mereo BioPharma), SEA-TGT (Seagen) y, HB0030 (Huabo Biopharma), AK127 (AKESO) or anti-TIGIT antibodies include Genntech antibodies (MTIG 7192A). In some embodiments, anti-TIGIT antibodies are as described in U.S. Pat. No. 9,713,364 (including MAB1、MAB2、MAB3、MAB4、MAB5、MAB6、MAB7、MAB8、MAB9、MAB10、MAB11、MAB12、MAB13、MAB14、MAB15、MAB16、MAB17、MAB18、40MAB19、MAB20 and/or MAB 21), anti-TIGIT antibodies are as described in U.S. Pat. No. 9,499,596, anti-TIGIT antibodies are as described in WO 2016/191643, anti-TIGIT antibodies are as described in WO 2017/053748, anti-TIGIT antibodies are as described in WO 2016/191643, anti-TIGIT antibodies are as described in WO 2016/028656, anti-TIGIT antibodies as described in WO 2017/030823, US2016/0176963, WO 2017/037707, WO 2017/059095, anti-TIGIT antibodies are as described in U.S.2017281764, anti-TIGIT antibodies are as described in WO 2015/009856, anti-TIGIT antibodies are as described in any one of US 2017/0037133, anti-TIGIT antibodies are as disclosed in WO 2017/048824 (including 10A7, 1F4, 14A6, 28H5, 31C6, 15A6, 22G2, 11G11 and/or 10D 7), the anti-TIGIT antibody is one of those antibodies described in international patent publication WO 2016/028656. In some embodiments, anti-TIGIT antibodies, typically full length or scFv domains, comprise the following CHA set of CDRs, the sequences of which are shown ：CPA.9.083.H4(S241P)vhCDR1、CPA.9.083.H4(S241P)vhCDR2、CPA.9.083.H4(S241P)vhCDR3、CPA.9.083.H4(S241P)vlCDR1、CPA.9.083.H4(S241P)vlCDR2 and cpa.9.083.h4 (S241P) vlCDR in fig. 30A. In some embodiments, anti-TIGIT antibodies, typically full length or scFv domains, comprise the following CHA set of CDRs, the sequences of which are shown ：CPA.9.086.H4(S241P)vhCDR1、CPA.9.086.H4(S241P)vhCDR2、CPA.9.086.H4(S241P)vhCDR3、CPA.9.086.H4(S241P)vlCDR1、CPA.9.086.H4(S241P)vlCDR2 and cpa.9.086.h4 (S241P) vlCDR3 in fig. 30B. Such anti-TIGIT antibodies may be used in combination with the anti-IL 18-BP antibodies of the invention. Additional exemplary anti-TIGIT antibody sequences are shown in figure 34.

In some embodiments, the anti-IL 18-BP antibody is used in combination with one or more anti-PVRIG antibodies. Examples of anti-PVRIG antibodies include, for example, but are not limited to, cha.7.518.1.h4 (S241P), cha.7.538.1.2.h4 (S241P) and CHA.7.502、CHA.7.503、CHA.7.506、CHA.7.508、CHA.7.510、CHA.7.512、CHA.7.514、CHA.7.516、CHA.7.518.1.H4(S241P)、CHA.7.518、CHA.7.518.4、CHA.7.520.1、CHA.7.520.2、CHA.7.522、CHA.7.524、CHA.7.526、CHA.7.527、CHA.7.528、CHA.7.530、CHA.7.534、CHA.7.535、CHA.7.537、CHA.7.538.1.2.H4(S241P)、CHA.7.538.1、CHA.7.538.2、CHA.7.543、CHA.7.544、CHA.7.545、CHA.7.546、CHA.7.547、CHA.7.548、CHA.7.549、CHA.7.550、CPA.7.001、CPA.7.003、CPA.7.004、CPA.7.006、CPA.7.008、CPA.7.009、CPA.7.010、CPA.7.011、CPA.7.012、CPA.7.013、CPA.7.014、CPA.7.015、CPA.7.017、CPA.7.018、CPA.7.019、CPA.7.021、CPA.7.022、CPA.7.023、CPA.7.024、CPA.7.033、CPA.7.034、CPA.7.036、CPA.7.040、CPA.7.046、CPA.7.047、CPA.7.049 and cpa.7.050 as well as other antibodies in clinical studies, e.g., GSK4381562/SRF816 (GSK/Surface), NTX2R13 (Nectin Therapeutics), as disclosed in WO 2018/220446 A9. In some embodiments, the antibody sequence is from WO 201/6134333. In some embodiments, anti-PVRIG antibodies, typically full length or scFv domains, include the following CHA set of CDRs, the sequences of which are shown ：CHA.7.518.1.H4(S241P)vhCDR1、CHA.7.518.1.H4(S241P)vhCDR2、CHA.7.518.1.H4(S241P)vhCDR3、CHA.7.518.1.H4(S241P)vlCDR1、CHA.7.518.1.H4(S241P)vlCDR2 and cha.7.518.1.h4 (S241P) vlCDR3 in fig. 29A. In some embodiments, anti-PVRIG antibodies, typically full length or scFv domains, include the following CHA set of CDRs, the sequences of which are shown ：CHA.7.538.1.2.H4(S241P)vhCDR1、CHA.7.538.1.2.H4(S241P)vhCDR2、CHA.7.538.1.2.H4(S241P)vhCDR3、CHA.7.538.1.2.H4(S241P)vlCDR1、CHA.7.538.1.2.H4(S241P)vlCDR2 and cha.7.538.1.2.h4 (S241P) vlCDR3 in fig. 30B. Such anti-PVRIG antibodies can be used in combination with the anti-IL 18-BP antibodies of the invention. Fig. 36, 37 and 38 show additional exemplary anti-PVRIG antibody sequences.

B. Combination therapy for other cancers

The anti-IL-18 BP antibodies of the invention may be administered in combination with one or more other prophylactic or therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotective agents, immunostimulating agents, immunosuppressants, agents that promote blood cell proliferation, angiogenesis inhibitors, protein Tyrosine Kinase (PTK) inhibitors or other therapeutic agents.

In this context, "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide, alkyl sulfonates such as busulfan, isopropanolamine, and piposulfamine; an aziridine group, which is a group of aliphatic hydrocarbons, such as benzotepa, carboquinone, rituximab and uredept; ethyleneimine and methylamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; polyacetic acid (especially bullatacin and bullatacin ketone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL'); beta-lapachone; lapaol; colchicine (colchicine); betulinic acid; camptothecins (containing synthetic analogues topotecan)CPT-11 (irinotecan,) Acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; bromostatin; CC-1065 (including adoxolone, calzelone and bizelone analogues thereof); podophyllotoxin; podophylloic acid; teniposide; candidiasis cyclic peptides (specifically, candidiasis cyclic peptide 1 and candidiasis cyclic peptide 8); dolastatin; docamicin (comprising synthetic analogues KW-2189 and CB1-TM 1); elstuporin (eleutherobin); a podocarpine (pancratistatin); the stoichiometriol (sarcodictyin); cavernosum (spongistatin); Nitrogen mustards such as chlorambucil, napthalamus, cholesteryl phosphoramide, estramustine, ifosfamide, dichloromethyl diethylamine, mechlorethamine hydrochloride, melphalan, mechlorethamine, chlorambucil cholesterol, prednimustine, trefosfamide, uracil mustard; nitrosoureas such as carmustine, chlorourea, fotemustine, lomustine, nimustine and ramustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 and calicheamicin omega 1 (see, e.g., agnew, chem intl. Edit engl.,33:183-186 (1994)); Epothilones; And neocarcinomycin chromophores and related pigmentary protein enediyne antibiotic chromophores), aclacinomycin, actinomycin, amphotericin, azaserine, bleomycin, actinomycin, karabin, carminomycin, acidophilic, chromomycin, actinomycin D, daunorubicin, dithiin, 6-diaza-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and doxorubicin), epirubicin, vorubicin, idarubicin, marrubicin, mitomycin such as mitomycin C, mycophenolic acid, noramycin, valdecomycin, Olivomycins, pelomycin, pofeomycin (potfiromycin), puromycin, tri-iron doxorubicin, rodobixin streptozotocin, streptozotocin tuberculin-killing agent tuberculin-killing agent a step of; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid, methotrexate, pterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioxanthine, thioguanine; pyrimidine analogs such as ambcitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluorouridine; androgens such as carbosterone, drotasone propionate, cyclothioandrostanol, emaandran, testosterone; anti-epinephrine such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; Acetoglucurolactone; aldehyde phosphoramide glycosides; aminolevulinic acid; enuracil; amsacrine; a method of treating a patient with a tumor; a specific group; eda traxas; a phosphoramide; colchicine; deaquinone; ornithine difluoride; ammonium elegance; epothilones; eggshell robust; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids are used in the preparation of a pharmaceutical composition, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mo Pai darol; diamine nitroacridine; prastatin; egg ammonia nitrogen mustard; pirarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; psk.rtm. polysaccharide complex (JHS Natural Products, eugene, oregon); Carrying out a process of preparing the raw materials; rhizopus extract; a sirzopyran; germanium spiroamine; tenuazonic acid; triiminoquinone; 2,2',2 "-trichlorotriethylamine; trichothecenes (specifically T-2 toxin, wart (verracurin) a, cyclosporin a, and serpentine; uratam; vindesineDacarbazine; mannitol; dibromomannitol; dibromodulcitol; pipobromine; ganciclovir (gacytosine); arabinoside ("Ara-C"); thiotepa; taxanes, e.g. taxolBristol-Myers Squibb Oncology，Princeton，N.J.)、Albumin engineered paclitaxel nanoparticle formulations without polyoxyethylated castor oil (American Pharmaceutical Partners, schaumberg, ill.) and docetaxel @Rhone-Poulenc Rorer, antony, france); chlorambucil; gemcitabine6-Thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastinePlatinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristineOxaliplatin; aldehyde hydrofolate; vinorelbineNorxiaoling; eda traxas; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid; capecitabineA pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the foregoing, such as CHOP, abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; CVP, abbreviation for combination therapy of cyclophosphamide, vincristine and prednisolone; and FOLFOX, i.e. oxaliplatinIn combination with the abbreviations of 5-FU and calcium folinate treatment regimen.

In some embodiments, the chemotherapeutic agent is selected from the group consisting of platinum, oxaliplatin, cisplatin, paclitaxel (taxol), sorafenib, doxorubicin, sorafenib, 5-FU, and gemcitabine, irinotecan (CPT-11).

In some embodiments, the other therapeutic agent is an agent used in radiation therapy for the treatment of cancer. Thus, in some embodiments, the active agents described herein are administered in combination with one or more of platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides.

In some embodiments, the anti-IL 18BP antibody is combined with one or more inflammatory body activators. In some embodiments, the inflammatory body activator is a CD39 inhibitor. In some embodiments, the CD39 inhibitor is an anti-CD 39 antibody.

According to at least some embodiments, the anti-IL 18BP antibodies may be used in combination with any cancer treatment standard of care known in the art (as may be found, for example, on the world wide web cancer. Gov/cancertopics).

Examples

Example 1: expression of IL18 and IL18BP in tumor microenvironments

IL18-BP is a chelator of IL18 and results in inhibition of IL18 activity (Dinarello, et al, front. Immunol.,1:1-10 (2013.) thus, in order to effectively block IL18-BP, both the target IL18-BP of the antibody and IL18 need to be present in TME (tumor microenvironment). FIG. 4 shows expression of both IL18 (FIG. 4A) and IL18-BP (FIG. 4B), and demonstrates that both proteins are expressed in all TCGA tumors, except for pheochromocytomas and paragangliomas (TCGA tumor type abbreviations see Table 1; The expression of IL18 was somewhat restricted in a subset of these tumor types (the reference line at 1RPKM provided a level of expression below its background). The final target cells for free IL18 are leukocytes/lymphocytes (tomnaga, k. Et al International Immunology,12 (2): 151-160 (2000) and Senju, h. Et al, int J Biol sci, 14 (3): 331-340 (2018)), and these studies will target tumors that show higher immune presence as demonstrated by ifnγ inflammatory signatures (see, e.g., US2016/0312295 A1). As can be seen from fig. 5, both IL18 (fig. 5A) and IL18-BP (fig. 5B) are ubiquitous in all tumor subgroups with inflammatory tumors (high ifnγ signature values). Even in the lowest ifnγ subpopulation, significant expression of both proteins was detected. Since IL18 is dependent on secretion of inflammatory corpuscles, a signature of the core inflammatory corpuscle genes is generated. These genes are common to many types of inflammatory body activation signals (Chauhan, D. Et al Immunological Reviews,297:123-138 (2020)). The signature values were calculated as the average of the log10RPKM expression values for the genes listed in table 2. As shown in fig. 6A, the core inflammatory body signature is highly expressed in all TMEs and is generally more highly expressed in the inflammatory (high ifnγ signature) sub-population. This pattern is very similar to that of IL18 (fig. 5A), indicating that the mechanism of IL18 is present in all inflammatory (high ifnγ signature) tumors and most low ifnγ signature tumors, except LGG and PCPG. To confirm that IL18 and the inflammation small body core signature genes were indeed expressed in the same cells of TME, single cell data were used and cosine similarity between IL18, IL18-BP and the core inflammation small body genes, as well as additional upstream genes and IL18 receptor was calculated. In fig. 6B, a cosine similarity matrix of macrophages in NSCLC is presented, indicating that IL18 and the core inflammatory body signature are indeed present in the same cells. Similar results were obtained in additional tumor types (data not shown). In particular, among breast cancers, triple negative breast cancers are reported to have more inflammatory characteristics in about 30% of cases, in contrast to about 5% to 10% in hormone receptor positive types (Thomas, f. Et al Frontiers in Oncology,10:1-17 (2021)). Thus, we examined the expression of IL18 and IL18-BP in breast cancer by single cell specificity (raw data were taken from Bassez, A. Et al, nature Medicine,27:820-832 (2021)). In fig. 7A, it is shown that by the percentage of expressing cells and the average expression level, both IL18 and IL18-BP are more enriched in TNBC, flowing from the her2+ subpopulation and being least expressed in hormone receptor positive subpopulations, in particular in pre-treated samples. In this particular dataset, patients were sampled prior to treatment and then neoadjuvant therapy with either aPD1 or aPD1 with chemotherapy was administered by resecting the tumor (in a therapeutic biopsy). The authors measured the clonal expansion of T cells after treatment, which could be considered as an alternative measure to the treatment of aPD1 (Bassez et al, nature Medicine,27:820-832 (2021)). FIG. 7B demonstrates that non-expanded patients have lower IL18 baseline levels, while those patients have higher IL 18-BP. This may be an indicator of the potential role of IL18-BP in attenuating IL18 activity and in blocking Immune Checkpoint Blocking (ICB) therapeutic activity. Both genes were up-regulated after treatment with aPD 1. These observations reinforce the selection of more severe inflammatory indications of TNBC in general and TNBC in particular.

Materials and methods

Pretreatment, filtration and normalization

UMI was quantified using CELLRANGER.0.2 (10X Genomics) and the reference transcriptome GRCh 38. If not otherwise stated, the subsequent analysis is performed using "Seurat" (https:// satijalab. Org/seurat /).

Clustering and cell type annotation

The first 15 principal components are used to construct the SNN graph and UMAP embeddings. The cellular annotation of Thomas et al 2021 is based on metadata submitted by the author.

Cells were clustered by nested PCA. Clusters are annotated by their gene signature expression.

The cosine similarity matrix of the desired genome is calculated by calculating the values of equation (i) for each pair of genes and their expression vectors.

Ifnγ inflammation signatures (as described in US2016/0312295 A1, the entire contents of which are incorporated herein by reference) were calculated in three steps:

the IFNgamma_up signature was calculated as the average of the log10 RPKM expression values of the following genes ：CCR5、HLA-DRA、CXCL13、CCL5、STAT1、KLRK1、NKG7、CXCL9、LAIR1、LAG3、CXCR6、KLRD1、GZMA、PRF1、SIGLEC14、PTPN22、CD86、SLA、SIRPG、CD72、HAVCR2、PSTPIP2、SLAMF6、CD84、CD300LF、CD3D、IFNG、CXCL11、CD2、CTSZ、GZMB、IL2RG、CXCL10、LILRB4、PDCD1、CCL8、CIITA、CCL4、IGSF6、PTPRC、CLEC9A、CST7、MYLIP、ITGAL、CDH1、PSTPIP1、GZMK、HLA-E、CD3E、TAGAP、TNFRSF9

Ifnγ_down signature was calculated as the average of log10 RPKM expression values for the following genes:

CLEC3B、NR4A2、EEF1G、PIK3CA、TYRO3、CX3CL1、ING1、BST1、ACKR3、UBB、PPARG、PTEN、THY1、CLCA1、EFEMP1、GAS6、ITM2A、CD55、NFATC1、BCL6、RETNLB、PDCD4、TIMP3、CDO1、POLR1B、DDR1、F2R、CTSG、LILRA5、CX3CR1、TBP、CLEC1B、RGS16、PTPN13、IRF1、MON1B、CPD、PHACTR2、OAZ1、CASP3、IFI16、ITGA1、RPL19、CCR6、LTK、C10orf54、SLAMF1 And TNFAIP8L2

3. Finally, the ifnγ inflammation signature was calculated as the difference between the two signatures: ifnγ=ifnγ_up ifnγ_down.

Clustering and cell type annotation

The first 15 principal components are used to construct the SNN graph and UMAP embeddings.

TABLE 1TCGA tumor abbreviations

Table 2: inflammatory corpuscle signature.

Gene name	Synonyms (synonyms)	Enzyme Commission numbering (EC)
			CASP1	IL1BC、IL1BCE	3.4.22.36
CASP4	ICH2	3.4.22.57
			CASP5	ICH3	3.4.22.58
PYCARD	ASC、CARD5、TMS1
			GSDMD	DFNA5L、GSDMDC1、FKSG10

Example 2: IL-18BP is soluble immune checkpoint-RNA expression data

Upregulation of IL-18BP in TME-TCGA and GTEX

Fig. 47: expression of IL18BP transcripts in normal (green) or cancer (red) tissues from TCGA and GTEX databases. GBM, glioblastoma multiforme; HSNC squamous cell carcinoma of head and neck; KIRC, renal clear cell carcinoma; PAAD, pancreatic cancer; SKCM, cutaneous melanoma; STAD, gastric adenocarcinoma (< P0.01).

IL-18BP is expressed in an inhibitory myeloid cell population and is associated with PD-L1 in TME, indicating a mechanism of resistance

As shown in fig. 59A: IL-18BP is associated with PD-L1 at the RNA level (TCGA) in colon and breast cancers, suggesting a mechanism of resistance to immune activation in the Tumor Microenvironment (TME)

As shown in fig. 59B and 48: single cell RNA analysis of tumor infiltrating bone marrow cells, including tumor-associated macrophages (TAMs) and Dendritic Cells (DCs) in colon cancer patients, showed up-regulation of IL-18BP in bone marrow cell populations in TME compared to Peripheral (PBMC), indicating a resistance mechanism to immune activation in TME.

As shown in fig. 59C: single cell RNA analysis of tumor infiltrating bone marrow cells, including tumor-associated macrophages (TAMs) and Dendritic Cells (DCs) in different indications, showed up-regulation of IL-18BP in bone marrow cell populations in TME compared to Peripheral (PBMC), suggesting a resistance mechanism to immune activation in TME

Up-regulation of IL-18BP in response to ICB treatment-scRNA/bulk RNA data

Fig. 60A to 60C: IL-18BP up-regulation (RNA levels) following Immune Checkpoint Blockade (ICB) treatment, IL-18BP levels up-Regulated (RNA) in the tumor microenvironment following treatment with anti-PD-1 (breast and basal cell carcinoma) or anti-PD-1 plus anti-CTLA-4 (melanoma), indicating a potential resistance mechanism.

Fig. 60D: the rise in IL-18BP in serum of NSCLC patients following treatment with agd- (L) 1 quantifies plasma IL-18BP protein levels at baseline by ELISA for healthy donors (n=22) and NSCLC patients (n=52) prior to treatment and after receiving treatment with anti-PD- (L) 1 (n=52) at subsequent CT scans.

Correlation of IL-18BP levels with adverse responses to aPD- (L) 1 blockade: 1) RCC (combination of pembrolizumab+lenvatinib) and 2) IL18BP in melanoma responders/NR (Olink)

IL-18BP is supportive data of soluble ICP and the action of potential resistance mechanisms to PD1 blockade in renal cell carcinoma patients receiving Pem Shan Kangjia lenvatinib. As can be seen in fig. 61A: high IL-18BP in serum of patients pre-treated with pembroliquanib Shan Kangjia was associated with a shorter Progression Free Survival (PFS). As can be seen in fig. 61B: high IL-18BP associated with stable or progressive disease (SD/PD) in patient serum was pre-treated with pembro Shan Kangjia lenvatinib.

IL-18BP is supportive data on soluble ICP and the action of potential resistance mechanisms to PD1 blockade in melanoma cancer patients receiving anti-PD-1 treatment. As can be seen in fig. 62, high IL-18BP in serum of melanoma cancer patients pretreated with anti-PD-1 was associated with adverse responses. After gray scale normalization of the target product, raw Olink data (NPX format) student T-test was performed on IL18BP proteins.

Example 3: inflammatory body-induced cytokines such as IL-18 and IL-1b are enriched in TME.

Unlike other cytokines, inflammatory body-induced cytokines such as IL-18 and IL-1b are enriched in TME.

The method comprises the following steps:

tumors were cut into small pieces using a surgical knife according to the manufacturer's protocol and transferred to GENTLEMACS ^TM C tubes containing enzyme mixtures (Miltenyi Biotec) using a human tumor dissociation kit (Miltenyi Biotec). After dissociation, the sample was centrifuged at 300g for 5 min and the supernatant was collected and centrifuged at 3130g for 10 min. After centrifugation, the supernatant was collected again and divided into aliquots and stored at-80 ℃. On the day of the assay, samples were thawed at room temperature, then centrifuged at 14,000rpm for 10 minutes, and supernatants were collected for immediate use in ELISA or CBA using the following kit:

● Human IL18 ELISA kit (MBL 7620)

● Human Th1/Th2/Th17 cytokine cell Counting Bead Array (CBA) (BD 560484)

Human inflammatory cytokine cell Count Bead Array (CBA) (BD 551811)

Results:

Fig. 71A: IL-18 and IL-1b are inflammatory small body-derived cytokines with opposite effects in TME. Although IL-18 promotes T cell and NK cell activation and leads to antitumor activity, IL1b has a dual role and leads to a pro-tumor activity in all effects.

Fig. 71B: the dot plot shows cytokine levels in tumor derived supernatants measured in various indications. Each point represents a sample. The average is depicted by the short black line. All other cytokines except IL-1b and IL-18 were below the lower detection limit.

Example 4: IL18 and IL18BP protein levels in patient serum compared to healthy donors and under different indications

The method comprises the following steps:

Serum samples from healthy donors and cancer patients were thawed and the levels of IL18 analyte (total amount of IL18, IL18 BP) were measured by the following ELISA kit according to the manufacturer's protocol:

● Human IL18 ELISA kit (MBL 7620)

● Human IL18BP ELISA kit (R & D DBP 180)

Results:

Figure 56A levels of IL18 analyte in serum of patients for different indications. FIG. 56B shows a dot pattern of IL18 analyte in serum samples from individual patients or healthy donors. Statistical analysis was performed using t-test (double tail), P < 0.001. The significantly increased expression of IL-18 in serum from cancer patients compared to healthy donors demonstrates an increased IL-18 level in the periphery during malignancy.

Example 5: elevated IL-18 protein levels in serum from head and neck cancer patients with tongue tumor sites

The method comprises the following steps:

Serum samples from head and neck cancer patients were thawed and the levels of IL18 analyte were measured by the following ELISA kit according to the manufacturer's protocol: human IL18 ELISA kit (MBL, 7620) and human IL18BP ELISA kit (R & D DBP 180).

Results:

Fig. 63A to 63B: principal Component Analysis (PCA) showed that mainly tumor sites were separated between samples with high and low levels of IL-18. The location of tumors in the tongue is associated with high levels of IL-18 and low levels of IL18BP, as compared to other sites. FIG. 63C shows serum levels of IL-18 and IL18BP in individual patients in a dot plot of different tumor sites.

Example 6: plasma IL18BP and IL18 protein levels in NSCLC patients treated with anti-PD 1/anti-PD 1 chemotherapy

The method comprises the following steps:

Plasma samples from NSCLC patients were thawed and the levels of IL18 and IL18BP were measured by the following ELISA kit according to the manufacturer's protocol: human IL18 ELISA kit (MBL, 7620) and human IL18BP ELISA kit (R & D DBP 180).

Results:

Fig. 65: the plasma of NSCLC cancer patients was collected at baseline and after single dose administration of anti-PD-1 (Keytruda) (n=8) or single dose administration of chemotherapy + anti-PD-1 (n=14). After a few treatment cycles, clinical assessment of patient response (response/no response, R/NR) is performed after PET-CT scan.

The average plasma levels of IL18BP and IL18 measured at baseline were higher for patients responding to therapy (anti-PD-1 monotherapy or anti-PD-1 and chemotherapy combination) compared to non-responding patients (fig. 65A).

As shown in fig. 65B and 65D, patients who were not clinically responsive to anti-PD-1 monotherapy exhibited higher IL18 and IL18BP plasma levels compared to baseline levels, whereas IL18 and IL18BP levels were not significantly altered from baseline in patients who were responsive to anti-PD-1 monotherapy. In contrast, only patients with clinical response against PD-1+ chemotherapy combination showed higher IL18 and IL18BP levels compared to baseline (fig. 65C, fig. 65D).

Discussion:

NSCLC patients treated with anti-PD 1 (Keytruda) are most likely to express PDL1-CPS >50%, potentially indicating increased immune infiltration in TME and subsequent increased IFNg expression. Given that IL18BP is an IFNg-induced gene, this may indicate a potential immune resistance mechanism in patients treated with anti-PD-1 and support combined anti-IL 18BP and anti-PD 1 blockade to further increase the rationality of the patient's potential anti-tumor response. For patients receiving chemotherapy + anti-PD-1 combination treatment, PDL1-CPS <50% and tended to have larger tumors. Patients with clinical responses to anti-PD-1+ chemotherapy combinations may have increased infiltration of immune cells that secrete IL18 and subsequent induction of IFNg levels, potentially resulting in increased secretion of IL18 BP. The clinical anti-tumor response of these patients can be potentiated with anti-IL 18BP antibodies.

Example 7: IL18 and IL18BP protein levels in Tumor Derived Supernatants (TDS)

The method comprises the following steps:

Tumors were cut into small pieces using a surgical knife according to the manufacturer's protocol and transferred to GENTLEMACS ^TM C tubes containing enzyme mixtures (Miltenyi Biotec) using a human tumor dissociation kit (Miltenyi Biotec). After dissociation, the sample was centrifuged at 300g for 5min and the supernatant was collected and centrifuged at 3130g for 10 min. After centrifugation, the supernatant was collected again and divided into aliquots and stored at-80 ℃. On the day of the assay, samples were thawed at room temperature, then centrifuged at 14,000rpm for 10 minutes, and supernatants were collected for immediate use in ELISA using the following kit:

● Human IL18 ELISA kit (MBL 7620)

● Human IL18BP ELISA kit (R & D DBP 180)

Results:

IL-18 and IL-18BP were detected in TDS under various indications.

FIG. 57 shows a plot of IL18 and IL18BP in TDS samples from individual patients. Fig. 58 IL18 and IL18BP levels in patient TDS for different indications.

Example 8: IL18RA is expressed on TIL of TME and its expression on CD4 TIL is induced compared to the periphery

The method comprises the following steps:

Tumor samples were cut into small pieces with a scalpel and transferred to GENTLEMACS ^TM C tubes (Miltenyi Biotec) containing enzyme mixtures. After dissociation, the cells were filtered through a 70 μm filter. Single cell suspensions were seeded into 96-well V-bottom plates and antibody mixtures (abs) directed against CD16 (BioLegend), CD32 (Thermo Fisher) and CD64 (BioLegend) were used to block Fc receptors. After staining the immune population with anti-human IL18Ra or with its isotype control (BioLegend), cells were obtained on FACS Fortessa hemocytometer (BD Bioscience) after washing (1% BSA,0.1% sodium azide in PBS).

Results:

IL-18Ra expression was induced on tumor-infiltrating T cells, statistically significant on CD4+ T cells, and trending on CD8+ T cells, compared to matched PBMC.

Fig. 55A shows the expression of IL18Ra on CD8 ⁺ and CD4 ⁺ and NK TIL from dissociated human tumors of various cancer types. Each point represents a different tumor of an individual patient. Fold expression values were calculated by dividing the MFI of the target by the MFI of the relevant isotype control. (FOI). Mean and SEM are shown by scale. Figure 55b expression of IL18Ra on CD4 ⁺ and CD8 ⁺ T cells and NK cells from donor matched PBMC and TMEs. Statistical analysis using paired t-test (two-tailed), P <0.05; * P=0.0064

Example 9: co-expression of TIGIT and IL18RA in TME

Human tumor dissociation kit Milteny (according to manufacturer's instructions) was used to mechanically dissociate and enzymatically digest tumor samples. Single cell suspensions were stained with Zombie-Nir to exclude dead cells and with antibodies against CD45, CD3, CD4, CD8, CD56, TIGIT or IL18 Ra. Cells were obtained on a FACS Fortessa hemocytometer (BD Bioscience) and analyzed with FlowJo software (V10). Cell surface markers were used to detect the following immune populations: cd8 (cd3+cd8+), CD4 (cd3+cd4+), NK (cd3-cd56+), and NKT (cd3+cd56+).

The results are shown in FIG. 33, which presents a flow cytometer point plot showing the co-expression of IL18Ra and TIGIT on endometrial and colonic TME, CD 8T cells, CD 4T cells, NK and NKT cells. Co-expression of TIGIT and IL18Ra on the same cell suggests that targeting both pathways by combined administration of inhibitory anti-IL 18BP and anti-TIGIT antibodies may have beneficial effects.

Example 10: generation and characterization of custom Ab to Adimab LTD of human IL18-BP protein

Production of anti-IL 18-BP hIgG1-N297A Ab against human IL18-BP protein

Antigen preparation

The antigen was biotinylated using the EZ-Link Sulfo-NHS-biotinylated kit (Thermo Scientific, catalog number 21425).

The antigen was concentrated to about 1mg/mL and the buffer was changed to PBS before the 1:7.5 molar ratio of biotinylation reagent was added. The mixture was kept at 4C overnight before another buffer exchange was performed to remove free biotin in the solution. Biotinylation was confirmed by streptavidin sensor binding of the labeled protein on ForteBio.

Natural library selection

Eight natural human synthetic yeast libraries were propagated as described previously, each library having about 10 ⁹ diversity (see, e.g., Y.xu et al, PEDS26 (10), 663-70 (2013); WO 2009036379;WO 2010105256 and WO 2012009568).

For the first two rounds of selection, the bead sorting technique was performed using the MILTENYI MACS system, as described previously (see, e.g., siegel et al, J Immunol Methods 286 (1-2), 141-153 (2004)). Briefly, yeast cells (about 10 ¹⁰ cells/library) were incubated with 10nM biotinylated human IL18-BP-Fc fusion in wash buffer (phosphate buffered saline (PBS)/0.1% Bovine Serum Albumin (BSA)) at 30℃for 30min. After one wash with 40mL ice cold wash buffer, the cell pellet was resuspended in 20mL wash buffer and streptavidin microbeads (500 μl) were added to the yeast and incubated for 15 minutes at 4 ℃. Next, the yeast was precipitated, resuspended in 5mL wash buffer and loaded onto MILTENYI LS columns. After 5mL loading, the column was washed 3 times with 3mL of wash buffer. The column was then removed from the magnetic field and the yeast eluted with 5mL of growth medium and then grown overnight.

Yeast was precipitated, washed three times with wash buffer and incubated with 10nM biotinylated human IL18-BP-Fc fusion, 10nM biotinylated cyno IL18-BP-Fc fusion, 100nM human IL18-BP-Fc monomer, 100nM biotinylated cyno IL18-BP monomer or multispecific reagent (PSR) at 30℃to remove non-specific antibodies from the selection, some selections were also made to enrich IL 18-competitive antibodies by incubation with biotinylated human IL18-BP-Fc fusion pre-complexed to human IL18, the library was incubated with biotinylated PSR reagent diluted 1:10 for depletion of PSR, yeast were then washed twice with wash buffer and anti-human kappa-FITC (LC-FITC) (Southern Biotech, cat# 2062-02) and 1:500 diluted streptavidin-AF 633 (SA-633) (Life Technologies, cat# 21375) or 1:50 diluted exoprotein-phycoerythrin (EA-PE) (Sigma-Aldrich, cat# E4011), secondary reagent(s) as described previously (see, e.g., Y. Xu et al, PEDS26 (10), 663-70 (2013)) and after washing twice with ice-cold wash buffer, cell pellets were resuspended in 0.3mL wash buffer and transferred to screen-capped sorting tubes using a FACS ARIA sorter (BD Biosciences), and the sorting gate is determined to select antibodies with the desired characteristics. The selection round is repeated until a population with all the desired characteristics is obtained. After the last round of sorting, the yeast was plated and individual colonies were picked for characterization.

Antibody optimisation

Optimization of antibodies was then performed via light chain batch shuffling followed by introducing diversity into the heavy and light chain variable regions as described below. A combination of some of these methods is used for each antibody.

Light chain batch shuffling: heavy chains from natural products are used to prepare light chain diverse libraries. Selection was performed on these libraries as described above, i.e., with one round MACS and four rounds FACS. In different FACS selection rounds, the library is evaluated for e.g. PSR binding and affinity pressure by antigen titration. Sorting is performed to obtain populations with the desired characteristics. Individual colonies were picked from each final FACS selection round for sequencing and characterization.

CDRH1 and CDRH2 selection: CDRH3 and CDRH1 and CDRH2 variants of the individual antibodies with a diversity of about 10 ⁸ were recombined into a pre-prepared library and selected using one round MACS and four rounds FACS as described in the natural selection. For each FACS round, the PSR binding and affinity pressure of the library was observed and sorted to obtain populations with the desired characteristics.

CDRH3 and CDRL3 selection: an oligomer comprising CDRH3 and flanking regions on either side of CDRL3 was ordered from IDT. Each oligomer is hybridized to one or two amino acids in CDR3 via NNK diversity. The CDRH3 oligomer was recombined with the heavy chain FR1-FR3 variable region comprising a variant selected from the group consisting of CDRH1 and CDRH2, and the CDRL3 oligomer was recombined with the light chain FR1-FR3 variable region from the parent antibody, the combined library diversity being about 10 ⁸. One round MACS and four rounds of FACS were used for selection as described in natural selection. For each FACS round, the PSR binding and affinity pressure of the library was observed and sorted to obtain populations with the desired characteristics. For these selections, affinity pressure was applied by pre-incubating the antigen with the parent IgG for 30 minutes, and then applying the pre-compounded mixture to the yeast library for a period of time to equilibrate the selection. Higher affinity antibodies can then be sorted.

Antibody production and purification

Yeast clones were grown to saturation and then induced with shaking at 30℃for 48 hours. After induction, yeast cells were pelleted and the supernatant was harvested for purification. IgG was purified using a protein a column and eluted with acetic acid pH 3.5.

Size exclusion chromatography

Quick SEC analysis was performed on mAb produced by mammals using TSKgel SuperSW mAb HTP column (22855) at a rate of 0.4mL/min with a cycle time of 6 minutes/round. 200mM sodium phosphate and 250mM sodium chloride were used as mobile phases.

Dynamic scanning fluorometry

10UL of 20-fold Sypro Orange was added to 20uL of 0.2mg/mL to 1mg/mL mAb or Fab solution. The temperature of the sample plate was raised from 40C to 95C in 0.5C increments using an RT-PCR instrument (BioRad CFX96 RT PCR) and equilibrated for 2 minutes at each temperature. Negative values of the first derivative of the raw data are used to extract Tm.

Table 3: aggregation of SEC-HPLC (%) and Fab Tm by DSF (. Degree. C.) for optimized antibodies

Cloning	Fab Tm(DSF，℃)	SEC-HPLC, monomer%
			ADI-71663	68.0	93.9
ADI-71701	71.5	98.6
			ADI-71707	71.0	98.8
ADI-71709	71.5	97.6
			ADI-71710	71.5	97.9
ADI-71719	72.5	94.5
			ADI-71720	72.5	98.6
ADI-71722	73.0	95.9
			ADI-71728	73.0	95.0
ADI-71736	73.5	79.2
			ADI-71739	73.0	94.9
ADI-71741	73.5	99.4
			ADI-71742	74.0	91.3
ADI-71744	74.5	88.1
			ADI-71753	74.5	96.9
ADI-71755	74.5	92.9

The anti-IL 18-BP hIgG1 Ab assay included the following steps:

Affinity measurements of anti-human Ab against human IL18-BP-Fc protein and cynomolgus monkey IL18-BP-Fc protein by ForteBio Octet-Natural export

As previously described, octet affinity measurements are typically performed on Octet HTX (see, e.g., estep et al, mAb 5 (2), 270-278 (2013)). Briefly, forteBio affinity measurements were performed by on-line loading IgG onto the AHC sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100nM antigen for 3 minutes and then transferred to assay buffer for 3 minutes to make an off-rate measurement. All kinetics were analyzed using a 1:1 binding model.

SPR measurement

Surface plasmon resonance K _D measurements

Kinetic analysis was performed in an HBS-EP+ running buffer system (10mM HEPES pH 7.4, 150mM NaCl,3mM EDTA,0.05% surfactant P20) at 25℃using a Biacore 8K optical biosensor (Global LIFE SCIENCES Solutions USA, marlborough, mass.). The sample chamber was maintained at 10 ℃ for the duration of each experiment.

For the antibody capture experiments, goat anti-human Fc antibody (Jackson ImmunoResearch) was covalently coupled to flow cells 1 and 2 of CM5 sensor chip surface via standard amine coupling (1:1 edc: nhs) and then blocked with ethanolamine (1.0 m, ph 8.5). Antibodies (10.0 nM in running buffer) were injected (40 seconds, 10. Mu.L/min) onto flow cell 2. A series of IL18-BP-Fc monomers ranging in concentration from 27.0nM to 0.111nM (3-fold dilution in running buffer) were injected (300 seconds, 30. Mu.L/min) onto flow cells 1 and 2. The dissociation of IL18-BP-Fc monomer was monitored for 600 seconds or 5130 seconds. Several blank buffer samples were injected (300 seconds, 30 μl/min) onto flow cells 1 and 2 and used for reference surface subtraction. All surfaces (flow cells 1 and 2) were regenerated via two injections (20 seconds, 30. Mu.L/min) of 10mM glycine pH 1.5.

For biotinylated antigen capture Fab or whole ab experiments in solution, each experimental cycle starts with injecting (150 seconds, 2 μl/min) a 1:20 solution of biotin capture reagent (Global LIFE SCIENCES Solutions USA) in running buffer on flow cells 1 and 2. The biotinylated IL18-BP-Fc fusion (10.0 nM) was then injected (120 seconds, 1.0. Mu.L/min) on flow cell 2. After capturing the biotinylated IL18-BP-Fc fusion to the sensor surface, a series of full ab concentrations (12.5 nM-0.8nM, 2-fold dilutions) of Fab concentrations (24.3 nM-0.1nM, 3-fold dilutions) were injected (300 seconds, 30. Mu.L/min) onto flow cells 1 and 2. Dissociation of Fab or Ab was monitored for 600 seconds or 5130 seconds. Several blank buffer samples were injected (300 seconds, 30 μl/min) onto flow cells 1 and 2 and used for reference surface subtraction. Finally, a regeneration solution (6M guanidine HCl in 0.25M NaOH) was injected (120 seconds, 10. Mu.L/min) on flow cells 1 and 2 to prepare the sensor surface for another cycle.

For data processing and fitting, the sensorgram is tailored to include only association and dissociation steps. Subsequently, the clipped data was aligned using Biacore weight evaluation software version 3.0.11.15423, subtracted by the double reference, and then nonlinear least squares fit to the 1:1 binding model.

The results are shown in fig. 41 and 42.

Fig. 41A: anti-IL 18BP Fab-human IL18BP interactions; biacore image of 10min dissociation.

Fig. 41B: anti-IL 18BP Fab-human IL18BP interactions; biacore image of 85 min dissociation.

Fig. 41C: anti-IL 18BP Fab-cyno IL18BP interaction, biacore image of 10min dissociation.

Fig. 41D: anti-IL 18BP Fab-cyno IL18BP interaction, biacore image of 85 min dissociation.

FIG. 42 presents a table showing KD values for human/cyno anti-IL 18BP Fab-IL18BP interactions measured by Biacore.

MSD-SET K _D measurement

Equilibrium affinity measurements were performed as described previously (Estep et al, 2013). Solution Equilibration Titration (SET) was performed in pbs+0.1% IgG-free BSA (PBSF), where the antigen (biotinylated IL18-BP-Fc fusion) was kept constant at 50pM and incubated with Fab serially diluted 1.5 to 3 fold from 10nM to 500pM (experimental conditions dependent on the sample). Antibodies (20 nM in PBS) were coated on standard MSD-ECL binding plates overnight at 4℃or 30min at room temperature. The plates were then blocked with BSA and shaken at 700rpm for 30 minutes followed by washing with wash buffer (PBSF+0.05% Tween 20). SET samples were applied and incubated on the plate for 150 seconds, vibrated at 700rpm, and then washed once. The antigen captured on the plate was detected in PBSF by incubation on the plate for 3 minutes with 1000ng/mL sulfo-labeled streptavidin. The plate was washed once with wash buffer and then read on MSD Meso Sector S instrument using 1-fold read buffer T with surfactant. The percentage of free antigen was plotted as a function of antibody titrated in Prism and fitted to a quadratic equation to extract K _D.

The results are shown in fig. 43 to 45.

Fig. 43A: superposition of Fab-IL18BP MSD image (black) with human IL-18-IL18BP MSD image (green).

Fig. 43B: superposition of Fab-IL18BP MSD image (black) with Cyno IL-18-IL18BP MSD image (green).

ForteBio Octet epitope divides case

Epitope binning was performed using a standard sandwich format cross-blocking assay. Control anti-target IgG was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100nM human IL18-BP-Fc antigen, followed by a second anti-IL 18-BP antibody. Additional binding of the second antibody after antigen binding indicates unoccupied epitopes (non-competitor), whereas non-binding indicates epitope blocking (competitor).

Alphalisa competition assay

Anti-HIS tagged receptor beads (PERKIN ELMER AL C) were incubated with 2.5nM human or cyno IL18-BP His along with 2.5nM biotinylated human or cyno IL18 and 150nM IgG for 60 min. After this incubation, streptavidin donor beads (PERKIN ELMER 670002S) were added and incubated for an additional 30 minutes at room temperature. The samples were then read using PERKIN ELMER ENSPIRE a multi-template reader (PERKIN ELMER 2390). After excitation at 680nm, the sample was read at 615 nm. Competition was calculated using photon (PBSF only)/photon (antibody) ratio. For top binders, the assay was repeated using antibody dose titration (150 nm, 3-fold dilutions).

Affinity measurement of anti-human IL18-BP Ab against human IL18-BP monomeric protein and cynomolgus monkey IL18-BP-HIS tag protein by BiaCORE-optimized output

For biotinylated antigen capture Fab experiments in solution, each experimental cycle starts with injecting (150 seconds, 2 μl/min) a 1:20 solution of biotin capture reagent (Global LIFE SCIENCES Solutions USA) in running buffer on flow cells 1 and 2. The biotinylated IL18-BP-Fc fusion (10.0 nM) was then injected (120 seconds, 1.0. Mu.L/min) on flow cell 2. After capturing the biotinylated IL18-BP-Fc fusion to the sensor surface, a series of Fab concentrations (24.3 nM-0.1nM, 3-fold dilutions) were injected (300 seconds, 30. Mu.L/min) onto flow cells 1 and 2. Dissociation of Fab was monitored for 600 seconds or 5130 seconds. Several blank buffer samples were injected (300 seconds, 30 μl/min) onto flow cells 1 and 2 and used for reference surface subtraction. Finally, a regeneration solution (6M guanidine HCl in 0.25M NaOH) was injected (120 seconds, 10. Mu.L/min) on flow cells 1 and 2 to prepare the sensor surface for another cycle.

Blocking human IL18 by ELISA: IL18-BP interaction

The inhibition of IL-18 (R & D) binding by human IL18-BP-Fc fusion protein by anti-human IL18-BP Ab from Adimab was tested by ELISA. Human anti-IL 18-BP polyclonal antibody (R & D, catalog number AF 119) was coated on wells of high binding plates overnight (2.5. Mu.g/ml, 50. Mu.l/well volume) at 4 ℃. The coated plates were rinsed once with PBS and incubated with 250 μl of blocking buffer (PBS with 2.5% skim milk) for 2 hours at Room Temperature (RT). Anti-human IL18-BP Ab (1:2, 4. Mu.g/ml to 0.06. Mu.g/ml, 50. Mu.L/well) was pre-incubated with serial dilutions of 1nM human IL18-BP-Fc and 4nM biotinylated human IL18 mixed at room temperature for 1 hour at room temperature. The blocking buffer was removed and the plates were washed and incubated with 100 μl/well of protein mixture for 2 hours at room temperature. Plates were washed and incubated with streptavidin-HRP solution (Jackson; 50. Mu.L/well volume) for 1 hour at room temperature. Plates were washed 3 times with PBS-T, 1 time with PBS and incubated with TMB substrate solution (50 μl/well) at room temperature to allow signal generation. The HRP reaction was stopped by adding 1N HCl solution (50. Mu.L/well) and the absorbance signal at 450nm was read on a luminescence reader (EnSpire, perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.). Blocking was calculated as a decrease in the binding signal of biotinylated human IL-18 to IL18-BP-Fc protein in the presence of Ab compared to the binding signal in the presence of isotype control.

Rescue of free human IL18 from pre-compounded human serum and recombinant human IL18 by ELISA

Rescue of human recombinant IL18 pre-bound by human IL18-BP in human serum by ELISA test of anti-human IL18-BP Ab from Adimab. Human anti-IL 18 Mab (MOR 09464-N30K antibody Novartis patent US2014/O112915A 1) was coated on wells of high binding plates overnight (2.5. Mu.g/ml, 50. Mu.l/well volume) at 4 ℃. The coated plates were rinsed once with PBS and incubated with 250 μl of blocking buffer (PBS with 2.5% skim milk) for 2 hours at Room Temperature (RT). Serial dilutions of anti-human IL18-BP Ab (1:2, 5-0.078 μg/ml,50 μl/well) were mixed and incubated at 37 ℃ for 2 hours with human healthy donor serum (ISERS 50 Almog) spiked with 4ng/ml recombinant human IL18 (R & D) for 1 hour at room temperature. For the standard curve, serial dilutions of recombinant IL18 (1:2, 3ng/ml-0.05 ng/ml) were performed in blocking buffer. The blocking buffer was removed and the plates were washed and incubated with 100 μl/well of protein mixture for 2 hours at room temperature. Plates were washed and incubated with D045-6 biotin (1:1000 in 1% BSA PBS, 100 μl/well, R & D) for 1 hour at room temperature. Plates were washed and incubated with streptavidin-HRP solution (Jackson; 50. Mu.L/well volume) for 1 hour at room temperature. Plates were washed 3 times with PBS-T, 1 time with PBS and incubated with TMB substrate solution (50 μl/well) at room temperature to allow signal generation. The HRP reaction was stopped by adding 1N HCl solution (50. Mu.L/well) and the absorbance signal at 450nm was read on a luminescence reader (EnSpire, perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.). IL18 rescue% was calculated as IL18 in the presence of Ab compared to the binding signal in the presence of isotype control: addition of free IL18 detected by the total amount of IL18-BP complex.

Rescue of free cyno recombinant IL18 from pre-complexed cyno recombinant IL18-BP and recombinant cyno IL18 by ELISA

Rescue of cyno recombinant IL18 pre-bound by cyno recombinant IL18-BP by ELISA test of anti-human IL18-BP Ab from Adimab. Human anti-IL 18 Mab (MOR 09464-N30K antibody Novartis patent US2014/O112915A 1) was coated on wells of high binding plates overnight (2.5. Mu.g/ml, 50. Mu.l/well volume) at 4 ℃. The coated plates were rinsed once with PBS and incubated with 250 μl of blocking buffer (PBS with 2.5% skim milk) for 2 hours at Room Temperature (RT). The blocking buffer was removed and the plates were washed and incubated for 1 hour at 37℃with 100. Mu.l/well of a serial dilution of anti-human IL18-BP Ab (1:3, 10. Mu.g/ml-0.004. Mu.g/ml, 50. Mu.l/well) with preformed cyno IL18: IL18-BP complex (1 ng/ml rhesus IL18, R & D and 25ng/ml IL18BP-His, R & D; incubation at 37℃for 1 hour). Plates were washed and incubated with D045-6 biotin (1:2000 in 1% BSA PBS, 100 μl/well, R & D) for 1 hour at room temperature. Plates were washed and incubated with streptavidin-HRP solution (Jackson; 50. Mu.L/well volume) for 1 hour at room temperature. Plates were washed 3 times with PBS-T, 1 time with PBS and incubated with TMB substrate solution (50 μl/well) at room temperature to allow signal generation. The HRP reaction was stopped by adding 1NHCl solution (50. Mu.L/well) and the absorbance signal at 450nm was read on a luminescence reader (EnSpire, perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.). IL18 rescue% was calculated as IL18 in the presence of Ab compared to the binding signal in the presence of isotype control: addition of free IL18 detected by the total amount of IL18-BP complex.

Rescue of free human IL18 from pre-complexed human IL18-IL18-BP by IL18 HEK293 reporter cells

0.1Ng/ml human IL18 (R & D) was pre-incubated with cell culture media from SUIT2 INF-gamma treated cells (24 h, 1000U/ml) expressing high levels of IL18-BP in the presence of Adimab Ab or isotype control at 3. Mu.g/ml. 50K/well HEK293 reporter cells (Invivogen) were seeded in 96-well plates of test medium (DMEM high glucose, 10% FBS,1% pen-strep,1% glutamine) and 20ul of sample was added to each well. Cells were incubated in a CO ₂ incubator at 37℃for 20 hours. The next day, 20. Mu.l of the induced cell supernatant was added to 180ul of pre-warmed Quanti-Blue solution (Invivogen) in 96-well plates. Cells were incubated at 37 ℃ for 1 hour and SEAP levels were measured by OD650 reading. All samples were measured in duplicate. Blocking was calculated as an increase in free IL18 detection in the presence of Ab compared to the level of free IL18 in the presence of isotype control.

Results:

production of anti-human IL18-BP hIgG1

Adimab yeast natural library was used for 5 rounds of selection and one round of counter selection against multi-specific reagents to deplete non-specific antibodies using human IL18-BP fused to an igg1 Fc protein or cynomolgus IL18-BP-Fc protein (Adimab). Human IL18 was added on top of the human IL18-BP antigen and several rounds of enrichment were performed to block antibodies.

In the first screening, the bio-layer interferometry (BLI) technique was used to read the bio-sensor platform (ForteBioRED 384) Octet instrument 740 clones were isolated, sequenced and screened for binding to human IL18-BP in Kd order. Of 740 clones, 341 clones were unique and identified as positive binders to human IL18-BP, 266 antibodies had an affinity for human monomeric IL18BP of less than 100nM. Secondary screening for the first 341 Octet positive antibodies included affinity measurements for cynomolgus monkey IL18-BP or cynomolgus monkey IL18-BP-HIS fused to the igg1 Fc monomeric protein. 195 antibodies were human/cyno cross-reactive. Initial antibody binning in an Octet instrument using an sandwich method; however, this analysis does not distinguish between possible IL18 competitors and non-competitors. To overcome this, antibodies were binned in FACS using ligand competition. Individual clones were tested in the presence or absence of 10nM Hu IL18BP Fc of 100nm il 18. All clones selected showed competition for binding to IL18BP with IL18 (antibodies only represent bin 1 and all antibodies were ligand competitive).

Next, variable heavy regions from the naturally selected 341 unique clones were subcloned into a pre-prepared light chain shuffling library. The LCBS library was selected as described above, 3 rounds of selection were performed using human or cynomolgus IL18-BP antigen and one round of reverse selection was performed using PSR.

In the first screening, the bio-layer interferometry (BLI) technique was used to read the bio-sensor platform (ForteBioRED 384) Octet instrument, 1152 clones were isolated, sequenced and screened for binding to human IL18-BP in KD order. Of 1152 clones, 658 were unique and identified as positive binders to human IL 18-BP. The antibodies were ordered based on binding affinity to the human IL18-BP-Fc protein and the first 87 clones were selected for additional characterization and purified from the culture medium of yeast expressing cells using an affinity column. Secondary screening for the first 87 Octet positive antibodies included affinity measurements for cynomolgus monkey IL18-BP or cynomolgus monkey IL18-BP-HIS fused to the igg1 Fc monomeric protein. Antibodies were binned according to IL18-BP-Fc binding and competition with human IL 18. IL18-BP-Fc was competitively bound in AlfaLISA assay using 150nM purified hIgG 1. Based on all of the above, the antibodies were ranked and the first 16 antibodies were screened in AlfaLISA using dose titration of antibodies (150 nm, 3-fold dilution). The first 6 blocking human/cyno IL18-BP binders were selected for optimization.

Relevant CDRs from the first 6 parental clones were shuffled into a pre-prepared CDRH1 and CDRH2 library and 3 rounds of selection were performed using either human IL18-BP monomeric protein or cynomolgus monkey IL18-BP-HIS protein at Adimab. 79 unique clones were identified and screened in Octet for binding to monomeric human and cyno IL18-BP protein.

Relevant CDRs from 79 unique clones were used to create CDRH3 and CDRL3 diverse libraries. CDRH3/L3 libraries were panned using a pre-complex of 10nM IL18-BP monomer with 100nM parent IgG to obtain Koff-enriched clones. 47 unique clones were identified and purified from the culture medium of yeast expressing cells using an affinity column. Analysis of the first 47 antibodies included affinity measurements for cynomolgus monkey and human IL18-BP monomeric proteins (actet and Biacore). All 47 clones reached the Koff limit of detection for Octet at 85 minutes dissociation. The affinity of 47 clones for human and cynomolgus IL18-BP was measured by Biacore, 24 clones out of 47 reached the Koff limit of detection at 85 minutes of dissociation, as measured by Biacore for human IL18-BP, and 5 clones reached the Koff limit of detection at 85 minutes of dissociation, as measured by Biacore for cyno IL18-BP (fig. 8). Competition for binding to IL18-BP-Fc with human IL18 was performed in AlfaLISA assay using purified hIgG1 at 15nM (FIG. 9).

Blocking human and cyno IL18-IL18-BP interactions by ELISA

The blocking activity of the parent mAb against human IL18-BP was analyzed by ELISA. As shown in FIG. 10, anti-human IL18-BP Ab (1:2, 4 μg/ml-0.06 μg/ml) showed dose-dependent blocking effects compared to isotype control. Anti-human IL18-BP Ab (1:3, 10. Mu.g/ml-0.01. Mu.g/ml) showed a binding to cyno IL18: dose-dependent blocking effects of IL18-BP interactions (figure 11). The IC50 values of the anti-human IL18-BP Ab are shown in FIG. 12.

Rescue of free human/cyno IL18 from pre-complexed human or cyno IL18-IL18BP by anti-IL-18 BP Ab

ELISA assays were used to demonstrate the ability of anti-human IL-18BP affinity-matured mAbs to release human IL18 that binds to human IL-18BP and cyno IL18 that binds to a cyno IL18BP protein.

As shown in fig. 31, the anti-human IL18BP mAb was able to compare to isotype control from preformed human IL-18: human IL18 is released from the IL-18BP complex.

As shown in fig. 32, the anti-human IL18BP mAb was titrated (10 ug/ml, serial dilutions 1:3) from preformed cyno IL-18: the cyno IL18 is released from the IL-18BP complex.

IL18 HEK293 reporter cells were used to demonstrate the ability of mAbs against human IL18-BP to rescue the binding of IL18-BP protein to human IL 18. The addition of 30ng/ml anti-human IL18-BP Ab was able to recover the free IL18 of all test antibodies (FIG. 13).

Using SnapGene MUSCLE alignments, consensus sequences were generated using optimized sequences from each parental library. High affinity IL18BP binders from each parental lineage were aligned to corresponding germline sequences. The consensus sequence was generated at a threshold of > 90%.

The 66650 lineage (VH 1-03; VL-kappa-1-5) consensus sequences include:

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence E-A-S-S-L-E-S; and

The 66670 lineage (VH 1-69; VL-kappa-1-12) consensus sequence includes

● CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is G or Y and X2 is A or S; x3 is N, I or V

● CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F;

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

The 66692 lineage (VH 3-23, VL-kappa-1-12) consensus sequences include:

● CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

● CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

The 66716 lineage (VH 1-39; VL-kappa-1-12) consensus sequences include:

● CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

● CDR-L2 having the sequence A-A-S-S-L-Q-S; and

Example 11: IL-18BP was determined by KinExA and Biacore: affinity of anti-IL-18 BP Ab to IL18BP compared to IL-18

ADI-71739: IL-18BP relative to IL-18: IL-18BP was found in human, cyno and clone W19089C (Biolegend): affinity and characterization information in IL-18BP (KinExA) and mice (Biacore).

Kinetic exclusion assayEquilibrium binding affinity and kinetics between unmodified molecules in solution were measured. For affinity analysis, the equilibrium dissociation constant Kd is determined experimentally and reflects the strength of the binding interaction. The association rate Kon is also determined experimentally, while the dissociation rate koff is generally calculated based on the following equation: koff=kd x kon.

Kd analysis requires one interaction partner to be immobilized onto a solid phase, which is then used as a probe to capture the other interaction partner, the Constant Binding Partner (CBP). For each experiment, one of the binding partners was titrated against the background of CBP and allowed to equilibrate. The solution is then briefly exposed to the solid phase and a portion of the free CBP is captured. The captured CBP is then labeled with a fluorescent secondary molecule. The short contact time with the solid phase is less than the time required for dissociation of the preformed complex in solution, thus "kinetically excluding" competition between the solution and the binding partner of the solid phase titration. Since the solid phase only serves as a probe for free CBP in each sample, the solution equilibrium does not change during the KinExA measurement. The signal generated from captured CBP, which is proportional to the concentration of free CBP in the equilibrated sample, is used to determine the Kd value. The KinExA Pro software performs a least squares analysis on the measured data to fit the best solution of Kd and CBP activity to a curve representing 1:1 reversible bimolecular interactions. For each data point along the curve, the x-axis reflects the molar concentration of binding partner titrated, while the y-axis reflects the percentage of free CBP at that particular titrant concentration at equilibrium.

SPR affinity measurement of anti-IL 18BP binding to mouse IL18BP

Fab preparation: fab fragments were prepared from 1mg of anti-IL 18BP igg 1N 97A yeast raised antibodies using a Fab digestion kit (Pierce, cat# 44985). SDS PAGE gel analysis of purified Fab fragments was performed under reducing and non-reducing conditions

All experiments were performed in a Biacore T100 optical biosensor (Global LIFE SCIENCES Solutions USA, marlborough, mass.)

Capture chip preparation

Human Fc capture reagent (Cytyva, BR 1008-39) was covalently coupled to flow cells 3 and 4 of the CM5 sensor chip surface at 10. Mu.g/mL in pH 5 acetate buffer via standard amine coupling followed by a 6 min blocking step with ethanolamine (1.0M, pH 8.5).

Binding kinetics of mouse IL18BP to anti-IL 18BP Fab

For the in-solution antigen capture experiments, each experimental cycle started with 10ug/ml solution of mouse IL18BP-Fc or hIgG1 isotype control injected (60 seconds, 5. Mu.L/min) on flow cells 3 and 4, respectively. After capturing the mouse IL18-BP-Fc fusion or isotype control to the sensor surface, a series of Fab concentrations (300 nM-2.21nM, 2-fold dilutions) were injected (60 seconds, 30. Mu.L/min) onto flowcells 3 and 4. Dissociation of Fab was monitored for 900 seconds. Several blank buffer samples were injected (60 seconds, 30 uL/min) onto flow cells 3 and 4 and used for reference surface subtraction. Finally, a regeneration solution (10 mM glycine ph=1.5) was injected (60 seconds, 10 μl/min) on flow cells 3 and 4 to prepare the sensor surface for another cycle.

As shown in FIG. 52, ADI-71739 binds human and cyno IL-18BP (Kd about 2910 fM, kd about 208fM, respectively) with higher affinity than human and cyno IL-18 (Kd about 441fM, kd about 345fM, respectively). ADI-71739 binds mouse IL-18BP (Kd about 4 nM) with a lower affinity than IL-18 (Kd about 3.7 pM).

Example 12: comparison of IL-18 BP-Biochemical assay between commercial Ab and Adimab anti-IL-18 BP AB

The method comprises the following steps:

blocking hIL18BP-hIL-18 interactions by ELISA-IL18BP plate binding using a-hIL18BP Ab

This assay was used to identify anti-human IL18BP abs that inhibit the binding interaction between human IL18BP and its counterpart human IL-18. Commercial Ab anti-human IL18BP (clone No. W19089C, catalog No. 947703, biolegend), ADI-71739 and ADI-71722 were tested for inhibition of binding of human IL18BP protein to IL-18 by ELISA. IL18BP-Fc protein was coated on wells of high binding plates overnight (1. Mu.g/ml, 100. Mu.l/well volume) at 4 ℃. Plates were washed three times with PBS-T buffer (1X PBS pH 7.4,0.05% Tween 20) and incubated with 250. Mu.L of blocking buffer (2.5% skim milk in PBS) for 2 hours at Room Temperature (RT). The blocking buffer was removed and the plate was washed three times with PBS-T buffer. The plate-bound ligand was incubated with anti-human IL18BP Ab in PBS buffer containing 1% BSA in two serial dilutions (2.5. Mu.g/ml-0.019. Mu.g/ml, 100. Mu.L/well volume) for 1 hour at room temperature. The plates were washed once with PBS. The plate-bound ligand was incubated with a solution of human IL-18 (catalog number 9124-IL, R & D) in PBS buffer (1 ng/ml, 100. Mu.L/well volume) containing 1% BSA for 1 hour at room temperature. Plates were washed three times with PBS-T (PBS containing 0.05% Tween 20). Biotinylated anti-IL 18 detection antibody (catalog number D045-6, R & D) 1:1000 was added to 1% BSA in PBS buffer (100. Mu.L/well). It was incubated at room temperature for 1 hour and the plates were washed again. Peroxidase streptavidin (Jackson, cat. No. 016-030-084) was added 1:1000 to a PBS solution (100. Mu.L/well) containing 2.5% skim milk for 1 hour at room temperature. Plates were washed three times with PBS-T buffer (1X PBS pH 7.4,0.05% Tween 20). ELISA signals were generated in all wells by adding 50. Mu.L of TMB substrate and incubating for 1.45 min to generate the signal. The HRP reaction was stopped by adding 50. Mu.L of 1N HCl and the absorbance signal at 450nm was read on an illuminated reader-EnSpire (Perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.).

Competition ELISA using complexes of soluble IL18-IL18BP and anti-IL 18BP Ab

This assay was used to identify anti-human IL18BP abs that inhibit the binding interaction between human IL18BP and its counterpart human IL-18. Commercial Ab anti-human IL18BP (clone number 136007, catalog number MAB1191, R & D systems) and ADI-66716 were tested for inhibition of binding of human IL18BP protein to IL-18 by ELISA. Human IL18BP antibody (catalog number AF119, R & D) was coated on wells of high binding plates overnight (1 ug/ml, 100. Mu.l/well volume) at 4 ℃. Plates were washed three times with PBS-T buffer (1X PBS pH 7.4,0.05% Tween 20) and incubated with 250. Mu.L of blocking buffer (2.5% skim milk in PBS) for 2 hours at Room Temperature (RT). The blocking buffer was removed and the plate was washed three times with PBS-T buffer. The complex was preformed with 0.25nM human IL-18-BP (R & D, cat. 119 BP) and 3nM human IL-18 biotin (9124-IL, R & D) in PBS buffer with 1% BSA for 1 hour at 37 c. The complexes were incubated with anti-human IL18BP Ab (4 ug/ml-0.06ug/ml,100 μl/well volume in PBS buffer with 1% BSA serially diluted twice) for 2 hours at room temperature. Peroxidase streptavidin (Jackson, cat. No. 016-030-084) was added 1:1000 to a PBS solution (100. Mu.L/well) containing 2.5% skim milk for 1 hour at room temperature. Plates were washed three times with PBS-T buffer (1X PBS pH 7.4,0.05% Tween 20). ELISA signals were generated in all wells by adding 50. Mu.L of TMB substrate and incubating to generate the signal. The HRP reaction was stopped by adding 50. Mu.L of 1N HCl and the absorbance signal at 450nm was read on an illuminated reader-EnSpire (Perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.).

Results:

The blocking activity of AB-71739 and AB-71722 was compared to commercial antibodies against IL18BP Ab (clone number W19089C, biolegand). As seen in FIG. 53, the blocking effect of AB-71739 and AB-71722 was superior to that of the Biolegend Ab.

The affinity of the Biolegend antibody (catalog number 947703, clone number W19089C) for human IL18BP was measured by kinex a and found to be 63.8pM (see methods in example 11).

The blocking activity of ADI-66716 was compared to an anti-IL 18BP Ab commercial antibody (clone No. 136007, catalog No. MAB1191, R & D systems) in a soluble blocking ELISA assay. Figure 54 shows that ADI-66716 (right hand bar) has a better blocking effect than R & D antibodies.

The affinity of the R & D antibody (catalog No. MAB 1191) for human IL18BP was measured by Biacore and found to be 2.73 x 10-10M (fig. 76).

Example 13: functional assessment of anti-IL 18-BP Ab from Adimab Activity

The method comprises the following steps:

NK-based assays for functional assessment of anti-IL 18-BP antibodies

Human NK cells were thawed in RPMI 1640 with 20% FBS and washed once again with whole RPMI ((RPMI 1640, 10% FBS,1% glutamine, 1% penicillin-streptomycin solution) cells were then seeded in 96-well plates at 50k cells/well and examined for their ability to restore IL-18 activity in 5% CO ₂ incubator with a combination of rhIL-12 (10 ng/ml, R & D systems, 10018-1L/CF), rhIL-18 (3 ng or 10ng/ml, R & D systems, 9124-IL/CF) and rhIL-18BP-Fc chimeric protein (1. Mu.g/ml, R & D systems 119-BP) in 96-well plates after 30min, a decreasing concentration of anti-human IL-18BP mAb or related isotype control was added to the culture.

PBMC-based assays for blocking endogenous secreted IL-18BP

Human PBMC were thawed in RPMI 1640 with 20% FBS, washed once more with full RPMI (RPMI 1640,10% FBS,1% glutamine, 1% penicillin-streptomycin solution), and incubated in a T-75 flask at 37℃in a 5% CO ₂ incubator for 24 hours to allow recovery. Cells were then seeded at 200k cells/well in 96-well plates and incubated with a combination of rhIL-12 (10 ng/ml, R & D systems, 10018-1L/CF), rhIL-18 (33.3 ng/ml or 2ng/ml, R & D systems, 9124-IL/CF) and a decreasing concentration of anti-human IL-18BP mAb or isotype control. Cells and all added solutions were prepared in complete RPMI medium with a final volume of 150 μl/well. Plates were incubated at 37 ℃ in a 5% CO ₂ incubator for 24 hours, after which the supernatant was collected for ifny secretion assessment. IL-18BP secretion was confirmed by IL-18/IL-18BP complex ELISA (R & D Systems, DY8936-05, not shown). All tests were performed in triplicate and each was repeated with two donors (ADI 66716 and ADI 66692) and five donors of affinity matured antibodies.

CD69 expression

After 24 hours of incubation, NK cell pellet was collected, washed from residual medium with PBS and labeled with reactive dye (Zombie NIR) diluted 1:1000 in PBS for 15 minutes in the dark at room temperature. Cells were then incubated with Fc receptor blocking solution (Trustain Fcx, biolegend,2.5 μl/reaction) for 10min at room temperature. To detect cell surface expression of CD69, cells were incubated with PE-anti-human CD69 Ab (BioLegend, 1. Mu.g/ml) on ice for 30 minutes in the dark. The cells were then washed once and analyzed using a macsquar analyzer.

Cytokine secretion

To measure ifnγ secretion of cells, supernatants were collected 24 hours after stimulation and NK cells were tested by CBA human ifnγ kit (BD Biosciences, 558269) or PBMCs were tested by CBA human Th1, th2, th17 cytokine kit (BD Biosciences, 560484).

Data analysis and statistics

All FACS files were analyzed by FlowJo software. EC50 s were calculated using GRAPHPAD PRISM software, where applicable.

Results:

mAbs were analyzed for their ability to block mIL18-BP-mIL-18 interactions in NK-based in vitro assays

The function blocking activity of mabs against recombinant human IL18-BP was assessed by NK-based assays. As shown in fig. 14, the anti-human IL18-BP Ab was able to block recombinant IL-18BP and fully restore the IL-18 activity depicted by ifny secretion and CD69 expression in a dose-dependent manner compared to isotype control. EC50 values are in the one-digit and two-digit nM range.

MAbs were analyzed for their ability to block mIL18-BP-mIL-18 interactions in PBMC-based in vitro assays

The function blocking activity of mabs against endogenous human IL18-BP was assessed by PBMC-based assays. As shown in fig. 15, the anti-human IL18-BP Ab was able to block endogenous IL-18BP and resume ifnγ secretion in a dose-dependent manner compared to isotype control.

Example 14: functional assessment of anti-IL-18 BP Ab in T cell-based assays in combination with ICB

Soluble immune checkpoints up-regulated in TME in response to ifnγ.αil-18BP restore T and NK activity. This provides a mechanism proposed for anti-PD-1 resistance in patients with high levels of ifnγ.

Activity: in vitro- αIL-18BP restores T cell and NK cell activity. The in vivo activity of αil-18BP Ab was used to demonstrate tumor growth inhibition both as monotherapy and in combination with ICB.

MEL624:TIL assay for functional assessment of anti-IL 18-BP antibodies

The method comprises the following steps:

Human MEL624 cells were thawed and grown in DMEM containing 10% FBS, 1% glutamine, 1% penicillin-streptomycin solution and 1% HEPES buffer. Cells were then seeded at 75k cells/well in 96-well plates in assay medium (IMDM containing 10% human serum, 1% glutamine, 1% MEM eagle, 1% sodium pyruvate and 1% penicillin-streptomycin solution) and incubated in a 5% co ₂ incubator at 37 ℃ for 1 hour prior to co-culture with human tumor-infiltrating lymphocytes previously expanded using known melanoma antigens (TILs). Human TIL was thawed in complete TIL medium and 75k cells/well were co-cultured with MEL624 cells to generate 1:1 effector: target ratio. The CO-cultured cells were then treated with rhIL-18 (30 ng/ml) and rhIL-18BP (1. Mu.g/ml) in a 5% CO ₂ incubator at 37℃for 30 minutes to form IL-18: IL-18BP complex. After 30 minutes, anti-human IL-18BP mAb (ADI-71722, dose titration, 30 μg/ml-0.01 μg/ml, dilution factor 1:3) or related isotype control (hIgG 130 μg/ml) was added to the co-culture to examine their ability to restore IL-18 activity. Cells and all added solutions were prepared in complete assay medium with a final volume of 200 μl/well. Plates were incubated at 37 ℃ in a 5% CO ₂ incubator for 24 hours before the supernatant was collected for cytokine secretion assessment. All tests were performed in triplicate and each was repeated with four TIL donors.

Cytokine secretion

To measure cytokine secretion by cells, supernatants were collected 24 hours post-stimulation and tested by CBA human Th1, th2, th17 cytokine kit (BD Biosciences, 560484).

If applicable, the EC50 value is calculated using GRAPHPAD PRISM software.

Results:

By MEL624: the TIL assay was used to evaluate the function blocking activity of mabs against recombinant human IL 18-BP. As shown in fig. 49A-49B and 50, the anti-human IL18-BP Ab (ADI-71722) was able to block recombinant IL-18BP and fully restore IL-18 activity as depicted by ifnγ secretion in a dose-dependent manner compared to isotype control.

CMV recall assay for functional assessment of anti-IL 18-BP Ab as monotherapy and in combination with anti-PVRIG/anti-TIGIT/pembrolizumab

The method comprises the following steps:

Human MEL-624 overexpressing PD-L1 cells was thawed and loaded with CMV pp65 peptide (0.03 μg/ml). Cells were seeded at 100K/well in 96-well plates and incubated with a combination of rhIL-18 (30 ng/ml, R & D systems, 9124-IL/CF) and rhIL-18BP-Fc chimeric protein (2. Mu.g/ml, R & D systems 119-BP) in a 5% CO ₂ incubator at 37℃for 30 minutes to form an IL-18+IL-18BP complex. After 30 minutes, anti-human IL-18BP (ADI-71722), anti-human PVRIG, anti-human TIGIT (anti-TIGIT), anti-human PD1 (pembrolizumab) or related isotype control (hIgG 4) was added to the cultures to examine their ability to restore IL-18 activity. All antibodies were added to reach a final concentration of 10 μg/ml. After 30 minutes incubation with the antibody, thawed CMV-reactive T cells were added to the culture. Cells and all added solutions were prepared in complete IMDM (IMDM medium containing 10% human serum, 1% glutamine, 1% MEM eagle, 1% sodium pyruvate and 1% penicillin-streptomycin solution) with a final volume of 200 μl/well. Plates were incubated at 37 ℃ in a 5% co ₂ incubator for 24 hours, after which the supernatant was collected for ifny secretion assessment. All tests were performed in triplicate and each was repeated with three donors.

Results:

The function blocking activity of mabs against recombinant human IL18-BP was assessed by CMV recall assay. As shown in fig. 49C-49D and 51, the anti-human IL18-BP Ab was able to block recombinant IL-18BP and fully restore IL-18 activity, as depicted by ifnγ secretion. The combination of anti-IL-18 BP Ab with anti-PVRIG/anti TIGIT/pembrolizumab resulted in more ifnγ secretion, indicating a beneficial effect on T cell activation.

Example 15: functional assessment of anti-IL-18 BP Ab in T cell-based assays in combination with ICB

Whole blood assay

As shown in fig. 66, the anti-IL-18 BP antibody Ab-71709 showed no signs of systemic immune activation in ID either as monotherapy or in combination with nivolumab. Flow, ex vivo system simulating human blood circulation. Fresh whole blood was taken from six healthy volunteers and immediately transferred to the whole blood circulatory system. The test item was administered and the blood was set to circulate at 37 ℃ to prevent clotting. Blood samples collected at the 24 hour time point were subjected to hematology and flow cytometer parameter analysis and then processed into plasma for cytokine analysis. The anti-CD 52 antibody alemtuzumab is included as a reference antibody due to its controlled cytokine release in the clinic. In contrast to alemtuzumab, anti-IL-18 BP antibodies did not induce any signs of systemic immune activation as monotherapy or in combination with the anti-PD 1 antibody nivolumab, depending on the various readings employed.

In vitro study to test the effect of ADI-71739 on killing melanoma cells by human TIL

As shown in FIG. 67, the anti-IL 18-BP antibody ADI-71739 increased killing of melanoma cells by tumor-infiltrating lymphocytes. A schematic of the assay setup is shown in fig. 67A. MEL624 cells were co-cultured with human TIL pre-enriched with MART1 or gp100 peptide-specific clones. rhIL-18 (R & D systems,50 ng/ml) and rhIL-18BP (R & D systems, 1. Mu.g/ml) were added to the co-culture for 30 minutes to form the IL-18:IL-18BP complex prior to treatment with 10. Mu.g/ml ADI-71739 or isotype control. The co-cultures were monitored using an intucyte live cell imaging instrument for 72 hours. As shown in fig. 67B, the addition of IL-18 (grey) enhanced tumor cell killing, as exemplified by lower confluence (left) and increased apoptosis (right) of MEL624 cells over time. IL-18BP abrogated the effects of IL-18 in the presence of isotype control antibodies (black), while anti-IL-18 BP antibodies (blue-green) were able to fully restore these effects.

In vitro study to test the effects of ADI-71739Ab in combination with other checkpoint blocking antibodies

As shown in FIG. 68, ADI-71739 increased CMV-specific T cell secretion IFNg both as monotherapy and in combination with aPVRIG/aTIGIT/pembrolizumab. A schematic of the assay setup is shown in fig. 68A. MEL624 cells overexpressing PD-L1 were loaded with CMV peptide pp65. Cells were incubated with rhIL-18 (R & D systems,30 ng/ml) and rhIL-18BP (R & D systems, 2. Mu.g/ml) for 30min to form IL-18:IL-18BP complex, and then cells were treated with 10. Mu.g/ml ADI-71739 or aPVRIG (anti-PVRIG) or aTIGIT (anti-TIGIT) or pembrolizumab (anti-PD-L1) or isotype control as monotherapy or in various combinations. CMV-specific T cells were then added to the culture and IFNg secretion was measured after overnight incubation. As shown in FIG. 68B, ADI-71739 alone was able to increase IFNγ secretion by T cells, and this effect was enhanced when combined with pembrolizumab/aPVRIG/aTIGIT.

In vitro study to test the effect of ADI-71739 on human TIL function in the Presence of endogenous IL-18BP levels

As shown in FIG. 69, the anti-IL 18BP antibody ADI-71739 increased release of IFNg by tumor-infiltrating lymphocytes. A. Schematic of the assay setup. MEL624 cells were co-cultured with human TIL pre-enriched with MART1 or gp100 peptide-specific clones. IL-18 (3.7 ng/ml) was added to the co-culture along with 5. Mu.g/ml ADI-71739 or isotype control. The co-culture was left for 18 hours, after which the IFNg level in the supernatant was measured. B. The level of IFNg in co-cultures treated with ADI-71739 (turquoise) was increased compared to isotype-treated samples (black). Representative examples from two TIL donors are shown.

The level of bound IL-18 in TME is greater than the amount required for T cell activation in vitro

The method comprises the following steps:

human MEL624 cells were thawed and grown in DMEM containing 10% FBS, 1% glutamine, 1% penicillin-streptomycin solution and 1% HEPES buffer. Cells were then seeded at 75k cells/well in 96-well plates in assay medium (IMDM containing 10% human serum, 1% glutamine, 1% MEM eagle, 1% sodium pyruvate and 1% penicillin-streptomycin solution) and incubated for 1 hour at 37 ℃ in a 5% co2 incubator before co-culturing with human tumor-infiltrating lymphocytes previously expanded using known melanoma antigens (TILs). Human TIL was thawed in complete TIL medium and 75k cells/well were co-cultured with MEL624 cells to generate 1:1 effector: target ratio. The co-cultured cells were then treated with rhIL-18 (1.23 ng/ml-300 ng/ml). Cells and all added solutions were prepared in complete assay medium with a final volume of 200 μl/well. Plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 hours before collecting supernatant for cytokine secretion assessment. All tests were performed in triplicate.

● Human IL18 ELISA kit (MBL 7620)

● Human free IL18 detection kit (internal scheme)

Human free IL18 ELISA protocol:

Anti-human IL18 hIgG1 clone 12GL (patent US2014/0004128A 1) was diluted to 1. Mu.g/ml in PBS and coated on ELISA plates overnight (100 ul/well) at 4 ℃. The coated plates were washed three times with PBST and incubated with 300 μl of blocking buffer (PBS with 1% BSA) for 2 hours at Room Temperature (RT). The blocking buffer was removed and the plates were washed three times with PBST. Human healthy donor serum was diluted 1:2 with PBS containing 1% BSA. The standard curve was generated by incubating 2-fold serial dilutions of human IL18 (starting from 1 ng/ml) in PBS containing 1% BSA. Plates were washed three times with PBST buffer (1XPBS pH 7.4,0.05% Tween 20) and 100 ul/well biotinylated anti-IL 18 detection antibody (catalog number D0456R & D; 1:1000 dilution in PBS containing 1% BSA). It was incubated for 1 hour and after antibody binding, the plates were washed again as described above. 100 ul/Kong Lagen peroxidase HRP-conjugated streptavidin, jackson,1:1000 was added and the plate incubated at room temperature for 1 hour, after which the antibody bound, the plate was washed again as described above. ELISA signals were generated in all wells by adding 50. Mu.L of TMB substrate (Scytek) and incubating for 5 to 20 minutes. The HRP reaction was stopped by adding 50. Mu.L of 1N HCL and the absorbance signal at 450nm was read (EnSpire, perkin Elmar). Assays were performed in duplicate. Data were analyzed using GRAPHPAD PRISM software.

Results:

a schematic of the assay setup is shown in FIG. 70A, with rhIL-18 (R & D systems,1.23-300 ng/ml) treated with thawed tumor-infiltrating lymphocytes (TILs) co-cultured with MEL624 cells at a 1:1 ratio for 24 hours. As can be seen in fig. 70B, rhIL-18 increased ifnγ secretion in a dose-dependent manner. rhIL-18 activates TIL at concentrations above about 1ng/ml and reaches saturation at about 100 ng/ml.

Fig. 70C: the levels of bound IL-18 in TDS for different indications were almost higher than required for T cell activation in vitro. The bound IL18 levels were calculated by subtracting free IL18 from total IL-18 measured for each sample by two separate ELISA kits. The red dotted line indicates the level required for functional activity (1.5 ng/gr). The black line indicates the median level of binding to IL-18 for each tumor type.

Example 16: generation and characterization of custom Ab to mouse IL18-BP protein

The method comprises the following steps:

Production of anti-mouse IL18-BP protein Fab

Fab used human combinatorial antibody library in AbD Serotec (Bio Rad, germany)The production service is cultured.The library is based on the human IgG1 Fab format, which consists of the first two domains of the antibody heavy and intact light chains.

Study design

Fab against mouse IL18-BP was generated in AbD Serotec (Bio Rad, germany). UsingPhage libraries were run using 3 rounds of enrichment and reverse selection against non-related human IgG1 fusion proteins to deplete non-specific antibodies, thereby raising antibodies against the mouse IL18-BP protein. Next, the enriched antibody Chi Ya from the phage display vector was cloned into an expression vector to determine the final Fab format. The Fab format selected was Fab-FH (monovalent Fab mini Ab, containing Flag and 6 His tags) using mouse IL18-BP Fc fusion protein, mouse IL18-BP fused to human IgG1 to culture antibodies.

Production of anti-mouse IL18-BP Fab

Fab generation at AbD Serotec includes the following steps:

1. Antigen immobilization-immobilization of antigen on a solid support. Standard methods use covalent coupling to the beads.

2. Phage display selection-panning-presentation on phage particlesThe platinum library is incubated with immobilized antigen. Nonspecific antibodies were removed by extensive washing and specific antibody phages were eluted by addition of reducing agents. Coli cultures were infected with eluted phage and helper phage to produce an enriched antibody phage library for the next round of panning. Typically, three rounds of panning are performed.

3. Subcloning into antibody expression vectors-after panning, the enriched antibody DNA is isolated as a pool and subcloned into Fab expression vectors. Coli was transformed with the ligation mixture and plated on agar plates. At this stage, each growing colony represents a monoclonal antibody.

4. Primary screening-colonies were picked and grown in 384 well microtiter plates. Antibody expression is induced and the culture is lysed to release the antibody molecules. Cultures were screened for specific antigen binding by ELISA.

5. Secondary screening-Koff sequencing of the first 95 ELISA-positive clones included in label-free dips and reads of the biosensor platform (ForteBioRED 384) using Biological Layer Interferometry (BLI) technique-hits from primary and secondary screening experiments were sequenced to identify unique antibodies.

6. Expression and purification-unique Fab was expressed and purified using one-step affinity chromatography.

7. Antibody QC-purified Fab was tested by ELISA using recombinant protein.

MAb Performance analysis

Affinity measurement of anti-mouse IL18-BPAb to mouse IL18-BP protein by ELISA

Mouse IL18-BP His fusion protein (Sino Biological) was coated overnight (2.5. Mu.g/ml, 50. Mu.l/well volume) on wells of high binding plates at 4 ℃. Mouse anti-histidine tag HRP was used to ensure IL18-BP His coating (diluted 1:500 in blocking buffer). The coated plates were rinsed once with PBS and incubated with 250 μl of blocking buffer (PBS with 2.5% skim milk—according to experimental instructions) for 2 hours at Room Temperature (RT). The blocking buffer was removed, the plate was rinsed once again with PBS and incubated with anti-mouse IL18-BP Ab from Biorad (1:3, 5. Mu.g/ml-0.002. Mu.g/ml, 50. Mu.L/well) for 2 hours at room temperature. Plates were washed 3 times with PBS-T (PBS containing 0.05% Tween 20), followed by 1 wash with PBS and incubation with HRP conjugated secondary antibody (50 μl/well) for 1 hour at room temperature. Plates were washed 3 times with PBS-T, 1 time with PBS and incubated with TMB substrate solution (50 μl/well) at room temperature to allow signal generation. The HRP reaction was stopped by adding 1N HCl solution (50. Mu.L/well) and the absorbance signal at 450nm was read on a luminescence reader (EnSpire, perkin Elmar). The data was exported to Excel (microsoft) and plotted in GRAPHPAD PRISM (GraphPad Software, inc.).

Affinity measurement of mouse IL18-BP protein by BiaCore

1. Immobilization of anti-human Fc: all SPR measurements were performed using a BiaCore T-100 instrument in PBS 0.05% Tween 25 running buffer. S series CM5 chip (catalog number BR100530 Cytiva) was primed in running buffer for 7 min. The chip was normalized with 8 min 70% glycerol injection. A mouse antibody capture kit (catalog No. BR100838 Cytiva) was used for capture. 0.4M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide in water was mixed with 0.1M N-hydroxysuccinimide in water at a 1:1 ratio and the chip surface was activated at 10 μl/min for 420 seconds. Next, 30 μg/ml mouse Fc capture reagent diluted in fixation buffer (10 mM sodium acetate pH 5.0, catalog No. BR100838 Cytiva) was injected at 5 μl/min onto all 4 channels until Δru reached 12000RU. The chip was blocked with 1M ethanolamine-HCl pH 8.5 at 10 μl/min for 7 min.

2. Capturing an anti-mouse IL18-BP antibody: for capture, AB-837 mIgG1D 265A (AbD 35328) was diluted to 10. Mu.g/ml in running buffer and injected through a specific channel at a rate of 5. Mu.l/min. CH1 was used to capture isotype control (SYNAGISMIGG D265A). When the capture level reached about 250RU, the injection was stopped.

3. Kinetic measurement of anti-mouse IL18-BP Ab: 12 parts of mouse IL18-BP-Fc (catalog number 122-BP, R & D) were injected at 30. Mu.l/min into all channels for 180 seconds in double serial dilutions diluted from 256nM in running buffer. The bound protein was monitored for dissociation from the captured antibody for 1000 seconds. After each cycle, the chip surface was regenerated with 10. Mu.l/min injection of glycine-HCl pH 1.7 for 60 seconds. The resulting sensorgrams were processed and double referenced using Biacore T100 evaluation software. Where appropriate, the sensorgram was fitted with a simple 1:1 kinetic binding model.

Blocking mIL18-BP-mIL-18 interaction by ELISA

The anti-human IL18-BP Ab from Biorad was tested for inhibition of the binding of the mouse IL18-BP fusion protein to IL-18 (Sino Biological) by ELISA. IL18-BP His fusion protein was coated on wells of high binding plates overnight (2.5. Mu.g/ml, 50. Mu.l/well volume) at 4 ℃. The coated plates were rinsed once with PBS and incubated with 250 μl of blocking buffer (PBS with 2.5% skim milk) for 2 hours at Room Temperature (RT). The buffer was removed and the plates were washed and incubated with serial dilutions (1:2, 5. Mu.g/ml to 0.04. Mu.g/ml, 50. Mu.L/well) of anti-mouse IL18-BP Ab from Biorad for 30 minutes at room temperature. Plates were washed and incubated with biotinylated mouse IL-18 in blocking buffer (1. Mu.g/ml, 50. Mu.L/well volume) for 1 hour at room temperature. The plates were washed and HRP conjugated secondary antibody was added for 1 hour (50 μl/well) at room temperature. ELISA signals were generated as described above. Blocking was calculated as a decrease in the binding signal of biotinylated mouse IL-18 to IL18-BP-His protein in the presence of Ab compared to the binding signal in the presence of isotype control.

In vitro blocking of mIL18-BP-mIL-18 interaction

Mouse CD3 ⁺ T cells were isolated from spleens of freshly harvested C57BL/6 mice using the easy Sep ^TM mouse T cell isolation kit according to the manufacturer's instructions and plated at 0.8 x 10≡6 cells/ml onto T-75cm ² flasks with anti-CD 3 coating (10 μg/ml). anti-CD 28 (1. Mu.g/ml) was supplemented and cells were cultured at 37℃for 3 days with 5% CO ₂. Cells were then harvested, washed and incubated in the presence of rmIL-12 (2 ng/ml) for an additional 24 hours. The following day, IL-18 and IL-18BP were complexed for 30 minutes at 37℃and 5% CO ₂ (25. Mu.l per well) and anti-IL-18 BP mAb (serial dilutions, 25. Mu.l per well) were added to the 96-well plates for an additional 30 minutes. Cells were harvested, washed, supplemented with rmIL-12 (0.1 ng/ml final) and added to IL-18/IL-18 BP/anti-IL-18 BP containing cells (40K/25. Mu.l/well) at 37℃and 5% CO ₂ for 24 hours. After 24 hours of incubation, supernatants were collected for ifnγ secretion analysis by mouse Th1/Th2/Th17 cell count bead arrays (CBA, BD Biosciences).

Results:

production of anti-mouse IL18-BP Fab

Panning was performed in 3 rounds of selection and counter selection against a non-relevant Fc-tagged control protein (recombinant mouse IL-15 ra Fc chimeric protein R & D, catalog No. 551-MR-100) using mouse IL18-BP fused to Fc of the hIgG1 protein in BioRad to deplete non-specific antibodies.

In the first screen, about 360 clones were examined for binding to mouse IL18-BP and non-related proteins by ELISA assay. Of the approximately 360 clones, 150 clones were identified as positive binders to mouse IL 18-BP. Secondary screening of the first 95 ELISA-positive clones included detection of the first three ELISA-positive clones in a label-free dipstick and read biosensor platform (ForteBioRED 384) using a Biological Layer Interferometry (BLI) technique, wherein abs are ordered based on their slowest rate. Confirmation screening by ELISA produced 41 positive unique Fab binders. Fab were purified and their binding to mouse IL18-BP was confirmed by ELISA. The 41 fabs were further analyzed by affinity measurement, blocking activity and binning (ELISA, data not shown) of the mouse IL18-BP protein. 11 fabs belonging to the same bin were identified, which showed high blocking and binding activity.

Fab was engineered into full length immunoglobulins by BioRad. The first 6 fabs (AbD 35357, abD35327, abD35346, abD35328, abD35350, abD 35344) were transformed into mouse IgG 1D 256A.

MAb Performance analysis

Affinity measurement of mouse IL18-BP protein by ELISA

The affinity of 6 BioRad purified mAbs to mouse IL18-BP was analyzed by ELISA. As shown in FIG. 16, anti-mouse IL18-BP, abD35328 (also referred to as "AB-837", "Ab 837") bound to the mouse IL18-BP His fusion protein (2.5. Mu.g/ml) with a Kd value of 0.4 nM.

SPR kinetic measurement of anti-mouse IL18-BP (AbD 35328)

The binding of AbD35328 to the mouse IL18-BP-Fc protein by the anti-mouse IL18-BP mAb is demonstrated in FIG. 17. For this interaction, the koff constant is below the detection limit of the instrument (< 10-91/S) and ka=4.93 x 10-4/M-S. The KD value cannot be uniquely determined and is estimated to be below 1 x 10-12M.

Analysis of mAb performance in functional blockade of mIL18-BP-mIL-18 interactions

Blocking activity of 6 BioRad purified mabs against mouse IL18-BP was analyzed by ELISA. As shown in FIG. 18, anti-mouse IL18-BP Ab (1:2, 5 μg/ml-0.04 μg/ml, PBS containing 2.5% skim milk) showed a dose-dependent blocking effect compared to isotype control. The IC50 value for anti-mouse IL18-BP (AbD 35328) was 3.3nM (FIG. 19).

MAbs were assayed for their ability to block mIL18-BP-mIL-18 interactions in an in vitro T cell activation assay

The function blocking activity of BioRad purified mabs against mouse IL18-BP was assessed in a T cell activation assay. As shown in fig. 20, anti-mouse IL18-BP Ab (AbD 35328) showed a dose-dependent blocking effect by enhancing ifnγ secretion compared to isotype control. The EC50 value against mouse IL18-BP was 7.9nM (FIG. 21).

Example 17: efficacy of anti-IL-18 BP as monotherapy and in combination with immune checkpoint blockers

This example describes the efficacy of anti-mouse IL18-BP mAb treatment as monotherapy or in combination with an immune checkpoint blocker in a CT26 murine colon adenocarcinoma model, a B16/Db-hmgp100 melanoma model, a MC38OVA ^dim CRC model, and an E0771 Triple Negative Breast Cancer (TNBC) model.

Materials and methods

Tumor challenge experiment:

CT26 colon cancer was purchased from ATCC (CRL-2638). Cells were cultured in RPMI 1640 (Biological Industries, 01-100-1A) containing 10% FBS (Biological Industries, 04-127-1A) and 100 μg/mL penicillin/streptomycin (Biological Industries, 03-031-1B). For tumor implantation, cells were harvested and washed, counted, suspended in cold RPMI 1640 and placed on ice. BALB/c mice ((female, 8 weeks) Envigo) were anesthetized with intraperitoneal injection of a mixture of 10% ketamine (Clorketam; SAGARPA Q-7090-053) and 10% xylazine (Sedaxylan; BE-V254834). Next, the backs of the mice were shaved and sterilized with 70% ethanol solution. Tumor cells were subcutaneously injected as 50 μl of 2.5X10 ⁵ CT26 cells into the right rear side of the mice.

B16/Db-hmgp cells were supplied by Dr.Hanada et al (HHS AGENCY) and licensed from NIH. B16/Db-hmgp100 cells were generated by double transduction of B16F10 with H-2Db and a retrovirus encoding a chimeric mouse gp100 consisting of human gp100 _25-33 and the remainder of mouse gp 100. Cells were cultured in RPMI 1640 (Biological Industries, 01-100-1A) containing 10% FBS (Biological Industries, 04-127-1A) and 100. Mu.g/mL penicillin/streptomycin (Biological Industries, 03-031-1B), 1% glutamine (Life technologies, 35050-038), 1% sodium pyruvate (Biological Industries, 03-042-1B), 0.01% 2-mercaptoethanol (Life technologies, 31350-010), 10. Mu.g/mL blasticidin (InvivoGen, ant-bl-05). 1X 10 ⁵ B16/Db-hmgp cells were subcutaneously injected into the right rear side of the mice.

In both CT26 and B16/Db-hmgp100 models, mAb administration was started on day 4 (monotherapy) or on day 7 (combination therapy) after tumor inoculation when tumor volume was 30mm ³ to 50mm ³; and was provided intraperitoneally (i.p.) at a final volume of 200 μl/injection for 3 weeks for a total of 6 administrations. Tumor growth was measured every 2 to 3 days with electronic calipers and recorded as 0.5x W2 XL mm ³. Mice were sacrificed with CO ₂ at study termination or at any of the following clinical endpoints: tumor volume is not less than 1800mm ³, tumor ulcer, weight loss is not less than 20% or dying appearance.

MC38OVA ^dim cells (clone number UC104H 10) were obtained from Peter MacCallum cancer center. Cells were grown in DMEM or RPMI medium containing 10% FBS, 1% glutamine, 1% sodium pyruvate, 0.01% 2-mercaptoethanol, 1% penicillin-streptomycin, 1% HEPES, 1% NEAA. MC38OVA ^dim cells (10 ⁶ or 1.2X10 ⁶) were subcutaneously injected into the right side of the mice at 50 ul/mouse. At a tumor volume of 130mm ³-260mm³, mice were randomly assigned to treatment groups. Mice were treated with 15mg/KG SYNAGIS isotype control or with AB-837mAb, twice weekly for 6 total treatments. Tumor growth was measured with an electronic caliper every 2 to 3 days and recorded as 0.5X W2X L mm3. The experimental endpoint was defined as a tumor volume of 1800mm ³. Mice that lost more than 10% of weight or 20% from the initial weight between measurements were excluded.

The E0771 murine TNBC model was purchased from CH3 BioSystems (product: #94A 001). Cells were cultured in RPMI 1640 (Biological Industries, 01-100-1A) containing 10% FBS (Biological Industries, 04-127-1A) and 100 μg/mL penicillin/streptomycin (Biological Industries, 03-031-1B). For tumor implantation, cells were harvested and washed, counted and suspended in 10 ⁷ cells/ml cold RPMI 1640 and placed on ice. C57BL/6 mice ((female, 8 weeks) Envigo) were anesthetized with an intraperitoneal injection of a mixture of 10% ketamine (Clorketam; SAGARPA Q-7090-053) and 10% xylazine (Sedaxylan; BE-V254834). Next, tumor cells were injected in situ into the right third mammary fat pad of C57BL/6 mice with a mixture containing 50. Mu.l of 5X 10 ⁵ E0771 cells and 50. Mu.l of Matrigel Matrix (Corning; 354234). At a tumor volume of 330mm ³, mice were randomly assigned to treatment groups with n=10. Mice were treated with 15mg/KG SYNAGIS D A isotype control, AB-837mIgG1-D265A, in combination with anti-PD-L1. mAb was injected twice weekly for 6 total treatments. Tumor growth was measured with electronic calipers every 2 to 3 days and recorded as 0.5X W2X L mm ³. The experimental endpoint was defined as a tumor volume of 1800mm ³. Mice that lost more than 10% of weight or 20% from the initial weight between measurements were excluded.

Antibody:

Phage display anti-mouse IL18-BP mAb (AbD 35328) engineered into mouse IgG 1D 265A isotype monoclonal antibody (mAb) used in this study was shown to bind IL18-BP and block binding of mll-18 to IL18-BP in an ELISA assay. The anti-mouse PD-L1 inhibitor on the mIgG1 scaffold used in this study was mAb yw243.55.s70 described in WO 2010/077634 (the heavy and light chain variable region sequences are shown in SEQ ID NOs 20 and 21 of WO 2010/077634, respectively), with the sequences disclosed.

All mabs were formulated in sterile PBS and endotoxin content was low (< 0.05 EU/mg).

Table 4. MAb was tested.

1	Mouse IgG1
			2	Mouse IgG 1D 265A
3	Reference anti-PD-L1 (mIgG 1)	YW243.55.S70
			4	Anti-IL 18-BP mAb mouse IgG 1D 265A	AbD35328
5.	Anti-PVRIG mAb 407 mAb mouse IgG1	BOJ-5G4-F4
			6.	Anti-TIGIT mAb mouse IgG1	CPA.9.086 M1

Study design for CT26 and B16/Db-hmgp model

Monotherapy

Female BALB/C (for CT 26) or C57BL/6 (for B16/Db-hmgp100 and E0771) mice of 6 to 8 weeks old were purchased from Envigo and acclimatized in SPF animal facilities for 1 week before starting the experiment. Mice were anesthetized, shaved and inoculated subcutaneously with 50 μl of 2.5x10 ⁵ CT26 or 1x10 ⁵ B16/Db-hmgp100 or 5x10 ⁵ E0771 cell tumor cells.

On day 4 post tumor inoculation, mice were treated with mabs (described in detail below) injected on days 4, 7, 11, 14, 18 and 21 post inoculation. Tumor growth was measured every 2 to 3 days with calipers.

Table 5. Treatment group.

Combination therapy

For the combination of anti-IL 18-BP with anti-mPD-L1 mAb treatment, mice were randomly assigned to the treatment group with n=10 as described below on day 7 post tumor inoculation. Mice were treated with mabs (described in detail below) injected at days 7, 11, 14, 18, 21 and 25 post tumor inoculation. For the combination of anti-IL 18-BP with anti-TIGIT or anti-PVRIG, anti-IL 18-BP, anti-PVRIG, anti-TIGIT and control SYNAGIS MIGG1-D265A (anti-IL 18-BP) and mIgG1 (anti-PVRIG, anti-TIGIT) were administered starting on day 4 post-inoculation. Mice were treated with mabs injected on days 4, 7, 11, 14, 18 and 21 after inoculation (as described in detail below).

Table 6. Therapeutic doses.

Study design of E0771 model

At a tumor volume of 330mm ³, mice were randomly assigned to treatment groups with n=10. Mice were treated with 15mg/KG SYNAGIS D A, 837mIgG1-D265A and in combination with anti-PD-L1 mAb, twice weekly for 6 total injections.

Table 7. Treatment group.

Table 8. Therapeutic doses.

Statistical analysis:

Two-way ANOVA was repeatedly measured, followed by repeated measurement of two-way ANOVA for the selected paired groups using JMP (Statistical Discoveries TM) software. Tumor growth measurements were analyzed by comparing tumor volumes measured on the last day of survival of all study animals. Statistical differences expressed as percent tumor free in mice were determined by the log rank Mantel-Cox test. A value of P <0.05 was considered significant. * p <0.05; * P <0.01; * P <0.001. The number of replicates and the number of animals per group performed for each experiment are depicted in the corresponding legend.

Results

Monotherapy Activity against IL18-BP and anti-mPD-L1 in an isogenic CT26 mouse tumor model

The effect of anti-IL 18-BP monotherapy in a mouse syngeneic CT26 tumor model was evaluated and compared to monotherapy with anti-mPDL 1. Mice were treated with isotype control antibody (mIgG 1 or mIgG1D 265A) or with anti-PD-L1 mIgG1 antibody (YW243.55.S70) or mIgG1D265A anti-IL 18-BP (mAb AbD 35328).

In a semi-therapeutic treatment model of CT26 colon cancer, monotherapy with anti-PD-L1 produced a similar tumor growth rate to treatment with the mIgG1 isotype control, although with statistically significant benefit to mouse survival. The group of mice treated with anti-IL 18-BP mAb as monotherapy showed similar tumor growth to mice treated with the mIgG 1D 265A isotype control with no survival benefit (figure 22).

Activity of anti-IL 18-BP and anti-PD-L1 combinations in isogenic CT26 mouse tumor models

Next, the efficacy of anti-IL 18-BP and anti-PD-L1 combination therapies in a mouse syngeneic tumor model was assessed.

In a therapeutic treatment model of CT26 colon cancer, administration of anti-PD-L1 and control mIgG 1D 265A isotype treatment starting at day 7 post-inoculation did not affect tumor growth, whereas the combination of anti-IL 18-BP mAb with anti-PD-L1 caused significant TGI (52%, p=0.03, fig. 22), translated a higher response rate (4 out of 10 individuals showed tumor volumes below 200mm ³) into statistically significant benefit to mouse survival (P <0.05, fig. 22) and promoted increased and sustained anti-tumor activity.

Monotherapy activity of anti-IL 18-BP in MC38OVAdim mouse tumor model

To confirm the antitumor activity of anti-mouse IL18BP, AB-837 as a single agent, 15mg/kg of AB-837 or isotype control was administered to mice vaccinated with MC38OVA ^dim tumor cells. Monotherapy with AB-837 produced 58% TGI (p <0.005, figure 78A).

Monotherapy Activity against IL18-BP and anti-mPD-L1 in a syngeneic B16/Db-hmgp mouse tumor model

We also studied the anti-tumor activity of AB-837 as monotherapy or in combination with anti-PD-L1 antibodies in less immunoinvasive mouse melanoma model B16/Db-hmgp 100.

Mice were treated with isotype control antibody (mIgG 1 or mIgG 1D 265A) or with anti-PD-L1 mIgG1 antibody (YW243.55.S70) or mIgG 1D 265A anti-IL 18-BP (mAb AbD 35328).

In the B16/Db-hmgp100 melanoma tumor model, monotherapy with anti-PD-L1 resulted in tumor growth rates similar to treatment with the mIgG1 isotype control, but did not benefit survival in mice. The group of mice treated with anti-IL 18-BP mAb as monotherapy showed a tumor growth inhibition of 31.4% compared to the mIgG 1D 265A isotype control, with a statistically significant trend (p=0.09, fig. 23). In additional experiments, treatment with IL18-BP mAb produced 54% TGI (p <0.005, FIG. 78B).

Activity of anti-IL 18-BP and anti-PD-L1 combinations in isogenic B16/Db-hmgp100 mouse tumor model

Next, the activity of anti-IL 18-BP and anti-PD-L1 combination therapies in B16/Db-hmgp100 mouse syngeneic tumor models was evaluated.

In the B16/Db-hmgp100 melanoma tumor model, administration of PD-L1 with control mIgG 1D 265A isotype control treatment started on day 7 post-inoculation resulted in 30.8% tumor growth inhibition compared to mice treated with isotype control (p=0.07, fig. 23). Whereas the combination of anti-IL 18-BP mAb with anti-PD-L1 elicited 31.1% TGI compared to anti-PD-L1 monotherapy, and significant TGI (52%, p=0.0023) compared to mice treated with isotype control treatment (fig. 23). Compared to the control group (P <0.05, fig. 23), the survival of mice treated with the combination of anti-IL 18-BP mAb and anti-PD-L1 alone reached a statistically significant increase.

Activity of anti-IL 18-BP and anti-TIGIT combinations in B16/Db-hmgp100 isogenic mouse tumor model

Next, we assessed the activity of anti-IL 18-BP and anti-TIGIT combination therapies in B16/Db-hmgp100 mouse syngeneic tumor models.

Mice treated with anti-IL 18-BP mAb or with anti-TIGIT mAb as monotherapy showed 34% (p= 0.0594) and 43% TGI (p=0.0105), respectively, compared to mice treated with isotype control (fig. 24). However, when mice were treated with a combination of anti-IL 18-BP and anti-TIGIT mAb, 35% and 24% TGI were shown, respectively, compared to anti-IL 18-BP and anti-TIGIT monotherapy. In addition, these mice exhibited 57% (p=0.02, fig. 24) of TGI compared to the group treated with isotype control mAb. This effect was also translated into a significant increase in survival in mice (p=0.013, fig. 24).

Activity of anti-IL 18-BP and anti-PVRIG combinations in B16/Db-hmgp100 isogenic mouse tumor model

The activity of anti-IL 18-BP and anti-PVRIG combination therapies in B16/Db-hmgp100 mouse syngeneic tumor models was also assessed.

Although mice treated with anti-IL 18-BP mAb monotherapy showed 34% (p= 0.0594) TGI, mice treated with anti-PVRIG had comparable tumor growth rates to mice treated with isotype control mAb (fig. 25). None of the monotherapy significantly improved survival in mice. However, when mice were treated with a combination of anti-IL 18-BP and anti-PVRIG mAb, they showed statistical significance (44%, p=0.0034) compared to mice treated with isotype control, further resulting in significantly improved survival of mice (p=0.0034) (fig. 25).

Anti-IL 18-BP Activity as monotherapy and in combination with anti-mPD-L1 in situ E0771 mouse tumor model

Monotherapy with anti-mouse IL18bp mAb,837mIgG1-D265A produced 83% TGI (P < 0.0001) in E0771 tumor bearing mice compared to synargis D265A isotype control (figure 72). The combined treatment of 837mIgG1-D265A with agd-L1 resulted in an enhanced response with a TGI of 61% compared to AB-837 administration as monotherapy (p=0.029) and a TGI of 91% compared to isotype control (fig. 72, p < 0.0001). These anti-tumor responses are translated into significant survival benefits: the combination of 837mIgG1-D265A with anti-PD-L1 significantly (p=0.004) increased survival of mice compared to monotherapy with 837mIgG1-D265A (p=0.01).

Conclusion(s)

The study described in this example assessed the in vivo anticancer efficacy of mAbs against IL18-BP as monotherapy or in combination with anti-PD-L1, anti-TIGIT or anti-PVRIG mAbs in 3 syngeneic mouse tumor models (CT 26, B16/Db-hmgp100 and E0771).

Treatment with 15mg/kg (300 μg/mouse) of anti-IL 18-BP as monotherapy in the minimal disease setting (i.e. starting treatment on day 4) resulted in an increase in TGI (0% -35%) in the CT26 and B16/Db-hmgp tumor models, but no statistically significant survival advantage. However, when anti-IL 18-BP mabs were administered in combination with anti-PD-L1, anti-TIGIT or anti-PVRIG therapies, synergistic effects were demonstrated by statistically significant tumor growth inhibition and increase in mouse survival.

In the E0771 tumor model, treatment with 15mg/kg (300 μg/mouse) of anti-IL 18BP Ab as monotherapy resulted in significant anti-tumor activity (83% TGI) compared to the control group. The activity of the anti-IL-18 BP Ab was further increased when administered in combination with anti-PD-L1 therapy.

In the MC38OVAdim tumor model, treatment with 15mg/kg (300 μg/mouse) of anti-IL 18BP Ab as monotherapy resulted in significant anti-tumor activity (58% TGI) compared to the control.

The in vivo effects of anti-IL 18-BP in combination with anti-mPD-L1 treatment were also demonstrated in MC38ova (data not shown).

Example 18: monotherapy with anti-IL 18BP mAb induces immunogenic memory in E0771 tumor model

This example describes the ability of an anti-IL 18-BP Ab to induce immunogenic memory.

Materials and methods

Tumor challenge experiment:

The E0771 murine TNBC model was purchased from CH3 BioSystems (product: #94A 001). Cells were cultured in RPMI 1640 (Biological Industries, 01-100-1A) containing 10% FBS (Biological Industries, 04-127-1A) and 100 μg/mL penicillin/streptomycin (Biological Industries, 03-031-1B). For tumor implantation, cells were harvested and washed, counted and suspended in 10 ⁷ cells/ml cold RPMI 1640 and placed on ice. C57BL/6 mice ((female, 8 weeks) Envigo) were anesthetized with an intraperitoneal injection of a mixture of 10% ketamine (Clorketam; SAGARPA Q-7090-053) and 10% xylazine (Sedaxylan; BE-V254834). Next, tumor cells were injected in a mixture containing 50. Mu.l of 5X 10 ⁵ E0771 cells and 50. Mu.l of Matrigel Matrix (Corning; 354234), in situ into the right third mammary fat pad of C57BL/6 mice. mAb administration was started on day 11 after tumor inoculation when tumor volume was 270mm ³; and was provided intraperitoneally (i.p.) at a final volume/injection of 200 μl for 3 weeks for a total of 6 administrations. Tumor growth was measured every 2 to 3 days with electronic calipers and recorded as 0.5x W2 XL mm ³. Mice were sacrificed with CO ₂ at study termination or at any of the following clinical endpoints: tumor volume is not less than 1800mm ³, tumor ulcer, weight loss is not less than 20% or dying appearance.

Tumor re-challenge experiment:

For the tumor re-challenge experiments, mice treated with anti-mouse IL18-BP mAb and age-matched female C57BL/6 mice were re-challenged with 5 x 10 ⁵ E0771 cells in the third breast fat pad on the left 90 days after primary E0771 inoculation (figure 27A). Tumor growth was monitored as described above.

Results:

anti-IL 18-BP and anti-mPD-L1 monotherapy Activity in isogenic E0771 in situ mouse tumor model

In the E0771 tumor model, mice were treated with an anti-PD-L1 mIgG1 antibody (YW243.55.S70) or an anti-IL 18-BP mIgG 1D 265A antibody (mAb AbD 35328).

In the in situ E0771 TNBC model, monotherapy with anti-PD-L1 resulted in 38.2% (p=0.72) tumor growth inhibition compared to treatment with the mIgG1 isotype control, with a benefit to mouse survival (p=0.0362). The group of mice treated with anti-IL 18-BP mAb as monotherapy showed 94.2% (p=0.0113) tumor growth inhibition compared to the mIgG 1D 265A isotype control (fig. 26). This anti-tumor response was translated into a statistically significant survival benefit (p=0.0011, fig. 26). When comparing the therapeutic effect against IL18-BP with the effect against PD-L1, a tumor growth inhibition of 81.6% was detected.

E0771 in situ tumor re-attack model for assessing the generation of immune memory

Since monotherapy with anti-IL 18-BP mAb induced complete rejection of E0771 tumors in mice, we examined whether this therapy induced immune memory production by re-challenge of mice without significant residual tumors (complete responders). Mice were treated with 15mg/kg of anti-IL 18-BP Ab or isotype control. Two months after primary tumor inoculation, 5x10 ⁵ E0771 tumor cells were again inoculated into mice without significant residual tumor and age-matched mice without tumor. 5 out of 10 naive tumor mice developed tumor progression, whereas none of the mice that completely rejected the primary tumor (5/5) developed tumors (fig. 27A, 27B, and 27C). In additional experiments, 8/9 of the re-challenged mice reject the tumor compared to 1/9 of the naive mice. The spleen weight of the re-challenged mice was significantly increased compared to the tumor naive mice (p=0.004, fig. 27D). Furthermore, we found a significant increase in the percentage of CD44 ⁺CD62L^-CD8⁺ effector T cells (22%, p=0.02, fig. 5E) and CD19 ⁺ cells (20%, p=0.04, fig. 27F) in the re-challenged mice compared to the tumor naive mice. No other statistically significant differences were detected. Taken together, these results indicate that anti-IL 18bp monotherapy induced systemic memory in the mouse E0771 model.

Example 19: administration of anti-IL 18BP is expected to have better therapeutic potential than engineered IL-18

Materials and methods:

Mouse antibodies and recombinant proteins

All mabs and recombinant proteins were formulated in sterile PBS and endotoxin content was low (< 0.05 EU/mg).

Cell culture:

9 days prior to inoculation, vials of MC38ovadim cells were thawed into RPMI medium containing 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% HEPES, 1% NEAA, 0.01% 2-mercaptoethanol, 1% penicillin-streptomycin. After centrifugation at 300Xg for 8 minutes, the cell pellet was resuspended, counted and seeded in a T175 flask. On days 7, 5 and 2, cells were isolated and re-seeded with 6-8 x 10 ⁶ cells/T175 flasks. On the day of inoculation, cells were isolated, centrifuged at 300Xg for 10 min, filtered through a 40. Mu.M cell filter, and resuspended in RPMI.

Mice were vaccinated:

Experiments were performed in C57Bl/6 (female, 6-8 weeks, envigo). Mice were anesthetized with an intraperitoneally injected mixture of 10% ketamine and 10% xylazine. Next, MC38ova cells (1.2X10 ⁶) were inoculated subcutaneously into the right side of the mice at 50 ul/mouse. Tumor growth was measured every 2 to 3 days with an electronic caliper and recorded as 0.5X W ² X L mm³.

Administering anti-mIL 18BP and engineered mIL-18 to tumor-bearing mice:

Mice were randomly assigned to treatment groups at a tumor volume of about 120mm ³ (day 9). Mice were treated with Synagis mouse IgG1, k isotype control 15mg/kg (IP), anti-mIL 18bp 837mIgG115mg/kg (IP), PBS (SC) or engineered IL-18 (SC) 0.32 mg/kg. The treatment groups were injected twice weekly for 6 total treatments. Tumor growth was measured every 2 to 3 days with calipers. Mice were weighed once a week. Mice were bled prior to treatment 4, 4 hours after treatment 4, and 24 hours after treatment 4. Serum was analyzed for the presence of IFNg, TNFa, MCP, IL6 by flow Cytomegalovirus (CBA) mouse inflammation kit (BD catalog No. 552364). Spleens were harvested from mice 24 hours after treatment 4 and weighed. For IL15 experiments, mice were treated with single doses of 0.5ug, 1.5ug IL15, or a mixture of 0.5ug IL15 and 2.33ug IL 15R.

Engineered IL-18 (also known as DR-18; sequences from U.S. patent publication 20190070262A1, listed therein as mCS2 (SEQ ID NO: 61)) showed NO binding IL18-BP：HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQSHHHHHH

Results:

Analysis of MC38ovadom tumor-bearing mice treated with anti-mIL 18bp and engineered mIL-18:

When mice were treated with anti-mIL 18bp, no weight loss was observed, similar to the control group (FIG. 73A). When serum from mice treated with anti-mIL 18bp was analyzed, no increase in inflammatory cytokines IFNg, TNFa, MCP and IL6 was observed. In contrast, mice treated with engineered mIL-18 had elevated serum IFNg, TNFa, MCP and IL6 levels 4 hours after treatment 4 and elevated serum IFNg levels 24 hours after treatment 4 (FIG. 73B). Mice treated with engineered mls-18 had very high serum levels of IL18 at 4 hours post-treatment (methods of IL18 detection also identified engineered IL 18), which returned to baseline 24 hours post-treatment 4 (fig. 73C). Spleens harvested from mice 24 hours after the 4 th anti-mIL 18bp treatment were similar to spleens harvested from mice in the control group, while spleens harvested from mice treated with engineered mIL-18 were 4.9 times greater than the average of the control group (FIG. 73D). Similarly, spleens harvested from mice treated with different concentrations of IL-15 were larger than the control group (fig. 73E).

Example 20: in murine tumor models, anti-IL-18 BP antibodies modulate tumor microenvironment without affecting the periphery

To further understand the effect of mouse anti-IL-18 BP Ab on tumor microenvironment and immune periphery, we studied immune compositions of tumors isolated from mice treated with Ab-837 monotherapy and compared to control groups treated with isotype control in MC38OVA ^dim tumors.

Materials and methods:

Mouse antibodies and recombinant proteins

Cell culture

9 Days prior to inoculation, vials of MC38OVA ^dim cells were thawed into RPMI medium containing 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% HEPES, 1% NEAA, 0.01% 2-mercaptoethanol, 1% penicillin-streptomycin. After centrifugation at 300Xg for 8 minutes, the cell pellet was resuspended, counted and seeded in a T175 flask. On days 7, 5 and 2, cells were isolated and re-seeded with 6-8 x 10 ⁶ cells/T175 flasks. On the day of inoculation, cells were isolated, centrifuged at 300Xg for 10min, filtered through a 40uM cell filter, and resuspended in RPMI.

Inoculation of mice

C57Bl/6 mice (females, 6-8 weeks, envigo) were anesthetized with an intraperitoneally injected mixture of 10% ketamine and 10% xylazine. Next, MC38OVA ^dim cells (1.2x10 ⁶) were inoculated subcutaneously into the right side of the mice at 50 ul/mouse. Tumor growth was measured every 2 to 3 days with an electronic caliper and recorded as 0.5X W ² X L mm³.

Administration of anti-mIL 18BP to phase-tumor-bearing mice

Mice were randomly assigned to treatment groups at a tumor volume of about 120mm ³ (day 9). Mice were treated with synnagis mouse IgG1, k isotype control 15mg/kg (IP), anti-mIL 18bp 837mIgG115mg/kg (IP). The treatment groups were vaccinated twice a week for a total of 4 treatments. Tumor growth was measured every 2 to 3 days with calipers. Mice were weighed once a week. Mice were bled prior to treatment 4, 4 hours after treatment 4, and 24 hours and 48 hours after treatment 4. Serum was analyzed for IL-18 (MBL, catalog number 7625) and IL18bp levels by ELISA (internal ELISA). Tumors were harvested from mice 24 hours after treatment 4.

Tumor immunophenotype of MC38OVAdim tumor microenvironment

Mice were vaccinated with MC38OVAdim and treated twice weekly with anti-mouse IL-18BP Ab or isotype control. Tumors, spleen and serum were collected. Tumor samples were divided into small pieces according to the manufacturer's protocol and transferred to GENTLEMACS ^TM C tubes (Miltenyi Biotec) containing enzyme mixtures using a human tumor dissociation kit (Miltenyi Biotec). After dissociation, the samples were centrifuged at 300g for 8 minutes and the supernatant was collected. The cells were filtered through a 70 μm filter. Single cell suspensions were seeded into 96-well V-bottom plates. Cell viability was labeled using a Zombie Aqua viability dye (BioLegend) and dead cells were excluded. To block fcγ receptors, cells were incubated with 10 μg/mL of anti-CD 16 and anti-CD 32 antibodies (BD Bioscience) in cold 1xPBS buffer for 10 min. Various immune populations were stained with anti-mouse antibodies (see table 9). For cytokine staining, cells were stimulated with 50ng/ml PMA, 1ug/ml ionomycin and BFA. Cells were then stained for extracellular membrane markers and intracellular cytokine expression. Cells were obtained on a FACS Fortessa hemocytometer (BD Bioscience). Analysis was performed using FlowJo. The collected supernatant was centrifuged at 3130g for 10 minutes. After centrifugation, the supernatant was collected again. The presence of IFNg in tumor supernatants and serum was analyzed by flow Cell Bead Array (CBA) mouse inflammation kit (BD catalog No. 552364).

Table 9: FACS staining plates for identifying immunophenotypes of different immune cell types in MC38ovadim tumor models.

Results:

When mice were treated with anti-mouse IL18BP, 41.1% inhibition of tumor growth was observed after 4 treatments (fig. 74). To evaluate the immune composition, tumors and spleens were harvested as described in materials and methods, single cell suspensions were generated, and cells were stained with the antibody set described in table 9. Tumor supernatants and serum were collected and analyzed for cytokine concentrations. Monotherapy with anti-IL-18 BP Ab resulted in an increase in the number of CD3 ⁺ (+100%, p=0.007) and CD8 ⁺ (+85%, p=0.0087) T cells in TME (fig. 75A-75C). The therapy also induced an increase in the concentration of IFNg ⁺ (+76), p= 0.0519) in the tumor supernatant (fig. 75D). When the activation markers were examined, CD8 ⁺CD69⁺CD107⁺ (+168%, p=0.0005) T cells (fig. 75E) and CD107 ⁺ (+54%, p=0.0094) NK cells (fig. 75F) were significantly increased. In the bone marrow compartment, the number of DC cells (+136%, p=0.0017) and the expression of MHC-II thereon (+40%, p=0.0138) were significantly increased (fig. 75G), indicating a potential increase in the ability to elicit T cells. No immune activation was observed when similar parameters were examined in spleen or serum of AB-837 treated animals. Only slight effects were detected-slight decrease in NK cells, macrophages and neutrophils, increase in CD69 expression on NK cells and decrease in mll 18Ra expression on NK cells (fig. 75H). IFNg was not detected in the sera of mice treated with anti-IL-18 BP Ab or isotype control (fig. 75I). In conclusion, monotherapy with AB-837 induced robust TME-suppressed immunomodulation without peripheral immune activation.

Example 21: efficacy of anti-IL-18 BP Ab in combination with chemotherapy

Method of

Antibody and oxaliplatin administration

C57Bl/6 mice (females, 6-8 weeks, envigo) were inoculated subcutaneously with 1.2X10. 10 ⁶ MC38OVA ^dim mouse tumor cells to the right at 50 μl/mouse. Tumor growth was measured every 2 to 3 days with an electronic caliper and recorded as 0.5X W ² X L mm³. The experimental endpoint was defined as a tumor volume of 1800mm ³. Mice that lost more than 10% of weight or 20% from the initial weight between measurements were excluded.

At a tumor volume of 110mm ³, mice were randomly assigned to two treatment groups: a group to which 5mg/kg oxaliplatin (Sigma-Aldrich, cat# 09512) was administered or a control group to which DDW was administered. When the tumor reached a volume of 140mm ³, the mice in each group were assigned to two separate groups: groups treated with 15mg/kg of anti-mouse IL18bp antibody or isotype control. Antibodies were injected twice weekly for 6 times a total treatment (see table 10 for details).

Table 10: experimental treatment group

Results

Combinations of oxaliplatin and anti-IL 18BP antibodies produce synergistic effects in MC38ova tumor models

To investigate the effect of combining anti-IL 18bp mAb with oxaliplatin in the MC38ovadim mouse tumor model, mice were grouped as described in table 10. As shown in fig. 77, administration of the combination therapy produced a synergistic anti-tumor response compared to monotherapy with a single drug. Combination therapy with oxaliplatin and anti-IL-18 BP mAb resulted in 72% TGI (p < 0.0001) compared to oxaliplatin monotherapy and 42% TGI (p=0.24) compared to anti-IL-18 BP mAb monotherapy.

***

The examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains.

All headings and chapter designations are used for clarity and reference only and should not be construed as limiting in any way. For example, those skilled in the art will recognize the usefulness of the various aspects from the different titles and chapters in accordance with the spirit and scope of the present invention as described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A composition comprising an anti-IL 18-BP (interleukin-18 binding protein) antibody for activating T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells and/or congenital lymphoid cells (ILCs) and/or modulating bone marrow cells for use in the treatment of cancer, wherein the antibody antagonizes at least one immunosuppressive effect of IL18-BP, optionally wherein the anti-IL 18-BP antibody blocks IL18: IL18-BP binding interaction, optionally wherein the anti-IL 18-BP antibody exhibits a binding affinity or KD of less than 1 pM.

2. A composition comprising an anti-IL 18-BP antibody, wherein the anti-IL 18-BP competes for binding with an antibody that binds to the human IL18-BP of SEQ ID No. 254 and/or the secretory chain of the human IL18-BP of SEQ ID No. 255 and/or competes for binding with IL 18.

3. A composition comprising an anti-IL 18-BP antibody, wherein the anti-IL 18-BP competes for binding with an antibody as described in US 8436148, WO 2019213686, WO 200107480, WO 2019051015, WO 2014126277A1, WO 2012177595, US 20140364341 and/or WO 2018060447.

4. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises: vhCDR1, vhCDR2, vhCDR, vlCDR1, vlCDR2, and vlCDR3 sequences selected from the group consisting of:

vhCDR1(SEQ ID NO:844)、vhCDR2(SEQ ID NO:845)、vhCDR3(SEQ ID NO:846)、vlCDR1(SEQ ID NO:847)、vlCDR2(SEQ ID NO:848) and vlCDR (SEQ ID NO: 849) sequences (66650) of FIG. 1I;

x. vhCDR1(SEQ ID NO:850)、vhCDR2(SEQ ID NO:851)、vhCDR3(SEQ ID NO:852)、vlCDR1(SEQ ID NO:853)、vlCDR2(SEQ ID NO:854) and vlCDR (SEQ ID NO: 855) sequences (66670) of FIG. 1J;

vhCDR1(SEQ ID NO:856)、vhCDR2(SEQ ID NO:857)、vhCDR3(SEQ ID NO:858)、vlCDR1(SEQ ID NO:859)、vlCDR2(SEQ ID NO:860) and vlCDR (SEQ ID NO: 861) sequences (66692) of FIG. 1K;

the vhCDR1(SEQ ID NO:862)、vhCDR2(SEQ ID NO:863)、vhCDR3(SEQ ID NO:864)、vlCDR1(SEQ ID NO:865)、vlCDR2(SEQ ID NO:866) and vlCDR (SEQ ID NO: 867) sequences (66716) of FIG. 1L;

vhCDR1(SEQ ID NO:65)、vhCDR2(SEQ ID NO:66)、vhCDR3(SEQ ID NO:67)、vlCDR1(SEQ ID NO:70)、vlCDR2(SEQ ID NO:71) and vlCDR (SEQ ID NO: 72) sequence (71719) of FIG. 2B;

xv. the vhCDR1(SEQ ID NO:75)、vhCDR2(SEQ ID NO:76)、vhCDR3(SEQ ID NO:77)、vlCDR1(SEQ ID NO:80)、vlCDR2(SEQ ID NO:81) and vlCDR3 (SEQ ID NO: 82) sequences of FIG. 2C (71720);

xvi. vhCDR1(SEQ ID NO:85)、vhCDR2(SEQ ID NO:86)、vhCDR3(SEQ ID NO:87)、vlCDR1(SEQ ID NO:90)、vlCDR2(SEQ ID NO:91) and vlCDR (SEQ ID NO: 92) sequences (71722) of FIG. 2D;

xvii. vhCDR1(SEQ ID NO:95)、vhCDR2(SEQ ID NO:96)、vhCDR3(SEQ ID NO:97)、vlCDR1(SEQ ID NO:100)、vlCDR2(SEQ ID NO:101) and vlCDR (SEQ ID NO: 102) sequence (71701) of FIG. 2E;

xviii. vhCDR1(SEQ ID NO:105)、vhCDR2(SEQ ID NO:106)、vhCDR3(SEQ ID NO:107)、vlCDR1(SEQ ID NO:110)、vlCDR2(SEQ ID NO:111) and vlCDR (SEQ ID NO: 112) sequence (71663) of FIG. 2F;

vhCDR1(SEQ ID NO:115)、vhCDR2(SEQ ID NO:116)、vhCDR3(SEQ ID NO:117)、vlCDR1(SEQ ID NO:120)、vlCDR2(SEQ ID NO:121) and vlCDR (SEQ ID NO: 122) sequences (71662) of FIG. 2G;

xx. the vhCDR1(SEQ ID NO:125)、vhCDR2(SEQ ID NO:126)、vhCDR3(SEQ ID NO:127)、vlCDR1(SEQ ID NO:130)、vlCDR2(SEQ ID NO:131) and vlCDR3 (SEQ ID NO: 132) sequences of FIG. 2H (66692);

xxi. vhCDR1(SEQ ID NO:135)、vhCDR2(SEQ ID NO:136)、vhCDR3(SEQ ID NO:137)、vlCDR1(SEQ ID NO:140)、vlCDR2(SEQ ID NO:141) and vlCDR (SEQ ID NO: 142) sequences of FIG. 2I (71710);

xxii. vhCDR1(SEQ ID NO:145)、vhCDR2(SEQ ID NO:146)、vhCDR3(SEQ ID NO:147)、vlCDR1(SEQ ID NO:150)、vlCDR2(SEQ ID NO:151) and vlCDR (SEQ ID NO: 152) sequences (71717) of FIG. 2J;

xxiii the vhCDR1(SEQ ID NO:155)、vhCDR2(SEQ ID NO:156)、vhCDR3(SEQ ID NO:157)、vlCDR1(SEQ ID NO:160)、vlCDR2(SEQ ID NO:161) and vlCDR (SEQ ID NO: 162) sequences (71739) of FIG. 2K;

xxiv the vhCDR1(SEQ ID NO:165)、vhCDR2(SEQ ID NO:166)、vhCDR3(SEQ ID NO:167)、vlCDR1(SEQ ID NO:170)、vlCDR2(SEQ ID NO:171) and vlCDR (SEQ ID NO: 172) sequences of FIG. 2L (71736);

xxv. vhCDR1(SEQ ID NO:175)、vhCDR2(SEQ ID NO:176)、vhCDR3(SEQ ID NO:177)、vlCDR1(SEQ ID NO:180)、vlCDR2(SEQ ID NO:181) and vlCDR (SEQ ID NO: 182) sequences of FIG. 2M (71707);

xxvi. vhCDR1(SEQ ID NO:185)、vhCDR2(SEQ ID NO:186)、vhCDR3(SEQ ID NO:187)、vlCDR1(SEQ ID NO:190)、vlCDR2(SEQ ID NO:191) and vlCDR (SEQ ID NO: 192) sequences (66716) of FIG. 2N;

xxvii. vhCDR1(SEQ ID NO:195)、vhCDR2(SEQ ID NO:196)、vhCDR3(SEQ ID NO:197)、vlCDR1(SEQ ID NO:200)、vlCDR2(SEQ ID NO:201) and vlCDR (SEQ ID NO: 202) sequences of FIG. 2O (71728);

xxviii. vhCDR1(SEQ ID NO:205)、vhCDR2(SEQ ID NO:206)、vhCDR3(SEQ ID NO:207)、vlCDR1(SEQ ID NO:210)、vlCDR2(SEQ ID NO:211) and vlCDR (SEQ ID NO: 212) sequences (71741) of FIG. 2P;

xxix. vhCDR1(SEQ ID NO:215)、vhCDR2(SEQ ID NO:216)、vhCDR3(SEQ ID NO:217)、vlCDR1(SEQ ID NO:220)、vlCDR2(SEQ ID NO:221) and vlCDR (SEQ ID NO: 222) sequences (71742) of FIG. 2Q;

xxx. vhCDR1(SEQ ID NO:225)、vhCDR2(SEQ ID NO:226)、vhCDR3(SEQ ID NO:227)、vlCDR1(SEQ ID NO:230)、vlCDR2(SEQ ID NO:231) and vlCDR (SEQ ID NO: 232) sequences of FIG. 2R (71744);

xxxi. vhCDR1(SEQ ID NO:235)、vhCDR2(SEQ ID NO:236)、vhCDR3(SEQ ID NO:237)、vlCDR1(SEQ ID NO:240)、vlCDR2(SEQ ID NO:241) and vlCDR (SEQ ID NO: 242) sequences (71753) of FIG. 2S; and

Xxxii. vhCDR1(SEQ ID NO:245)、vhCDR2(SEQ ID NO:246)、vhCDR3(SEQ ID NO:247)、vlCDR1(SEQ ID NO:250)、vlCDR2(SEQ ID NO:251) and vlCDR (SEQ ID NO: 252) sequences (71755) of FIG. 2T.

5. The composition of claim 1 or 2, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain of an antibody selected from the group consisting of

6. The composition of any one of claims 1 to 5, wherein the antibody comprises a CH 1-hinge-CH 2-CH3 region from human IgG1, igG2, igG3, or IgG4, wherein the hinge region optionally comprises a mutation.

7. The composition of any one of claims 1 to 6, wherein the antibody comprises the CH 1-hinge-CH 2-CH3 region from human IgG 4.

8. The composition of any one of claims 1 to 7, wherein the hinge region comprises a mutation.

9. The composition of any one of claims 1 to 8, wherein the antibody comprises the CL region of a human kappa 2 light chain.

10. The composition of any one of claims 1 to 9, wherein the antibody comprises the CL region of the human λ2 light chain.

11. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S-L-E-S; and

12. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

B) CDR-H2 having the sequence G-I-I-P-X-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is G or Y; x2 is A or S; x3 is N, I or V; and

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S

C) CDR-L3 having the sequence Q-Q-V-Y-X-X2-P-W-T, wherein X is S or R; x2 is L, I or F-.

13. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

a) CDR-H1 having the sequence F-T-F-X-N-X2-A-M-S, wherein X is G or D or S; x2 is T or V or Y;

b) CDR-H2 having the sequence A-I-S-X-X1-X2-G-S-T-Y-Y-A-D-S-V-K-G, wherein X is G or A; x2 is N or S; x3 is A or G; and

C) CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

C) CDR-L3 having the sequence Q-H-A-X-X1-F-P-Y-T, wherein X is Y or L; x1 is S or F.

14. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S

15. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S-L-E-S; and

16. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

b) CDR-H2 having the sequence G-I-I-P-G-X2-G-T-A-X3-Y-A-Q-K-F-Q-G, wherein X is any amino acid; x2 is any amino acid; x3 is any amino acid; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

17. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

C) CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y;

A light chain variable domain, the light chain variable domain comprises:

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

18. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

19. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence E-A-S-S- -E-S, wherein X is L or S; and

20. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

C) CDR-H3 having the sequence A-R-G-R-H-X-H-E-T, wherein X is S, G or F; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

21. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

C) CDR-H3 having the sequence A-K-G-P-D-R-Q-V-F-D-Y; and

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-X-S-W-L-A, wherein X is S or D

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

22. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

A light chain variable domain, the light chain variable domain comprises:

a) CDR-L1 having the sequence R-A-S-Q-G-I-S-S-W-L-A

B) CDR-L2 having the sequence A-A-S-S-L-Q-S; and

23. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

24. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

I) vlCDR1, vlCDR2 and vlCDR3 from VL-κ-1-5、VL-κ-1-12、ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755;

Wherein optionally, the CDR comprises 0 to 4 substituents, and wherein no individual CDR comprises more than 1 substituent, and wherein the vhCDR and vlCDR3 do not comprise substituents.

25. A composition comprising an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody comprises:

i) Comprising a heavy chain variable domain exhibiting a sequence having at least 90%, at least 95% or at least 98% identity to said heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vhCDR comprises no more than 1 substituent, and wherein said vhCDR3 comprises no substituents, and

Ii) comprises a light chain variable domain that exhibits at least 90%, at least 95%, or at least 98% identity to the light chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vlCDR comprises no more than 1 substituent, and wherein the vlCDR3 does not comprise a substituent.

26. A composition comprising an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody comprises:

i) Comprising the heavy chain variable domains of vhCDR, vhCDR and vhCDR3 from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and wherein the heavy chain variable domain comprises a sequence exhibiting at least 90% identity to the heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vhCDR comprises no more than 1 substituent, and wherein the vhCDR3 does not comprise a substituent, and

Ii) comprises the light chain variable domains of vlCDR, vlCDR and vlCDR from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and wherein the light chain variable domains comprise sequences that exhibit at least 90% identity to the light chain variable domains from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, wherein each individual vlCDR comprises no more than 1 substituent, and wherein the vlCDR3 does not comprise a substituent.

27. The composition of any one of claims 1 to 17, wherein the antibody comprises the heavy chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755, and the light chain variable domain from ADI-71663、ADI-71662、ADI-66692、ADI-71701、ADI-71709、ADI-71710、ADI-71707、ADI-71717、ADI-71719、ADI-71220、ADI-71722、ADI-71736、ADI-71739、ADI-71728、ADI-66716、ADI-71741、ADI-71742、ADI-71744、ADI-71753 or ADI-71755.

28. The composition of any one of claims 1 to 23, wherein the antibody comprises the CH 1-hinge-CH 2-CH3 region from human IgG 4.

29. The composition of any one of claims 1 to 24, wherein the hinge region comprises a mutation.

30. The composition of any one of claims 1 to 25, wherein the antibody comprises the CL region of a human kappa 2 light chain.

31. The composition of any one of claims 1 to 26, wherein the antibody comprises the CL region of the human λ2 light chain.

32. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

33. A composition comprising an anti-IL 18-BP antibody, wherein the antibody comprises:

Optionally, 1) wherein each CDR individually comprises 0 to 4 substituents, and wherein no individual CDR comprises more than 1 substituent, and wherein said vhCDR and vlCDR do not comprise substituents, 2) each CDR individually comprises 1 substituent, or 3) wherein each individual vhCDR comprises no more than 1 substituent, and wherein said vhCDR3 does not comprise a substituent.

34. The composition of any one of claims 1 to 33, wherein the antibody comprises a CH 1-hinge-CH 2-CH3 region from human IgG1, igG2, igG3, or IgG4, wherein the hinge region optionally comprises a mutation.

35. The composition of any one of claims 1 to 34, wherein the antibody comprises the CH 1-hinge-CH 2-CH3 region from human IgG 4.

36. The composition of any one of claims 1 to 35, wherein the hinge region comprises a mutation.

37. The composition of any one of claims 1 to 36, wherein the antibody comprises the CL region of a human kappa 2 light chain.

38. The composition of any one of claims 1 to 37, wherein the antibody comprises the CL region of the human λ2 light chain.

39. A composition comprising any one of the preceding claims, wherein the antibody competes for binding with an antibody according to any one of the preceding claims.

40. A method of treating cancer in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the cancer is treated.

41. A method of treating cancer in a patient, the method comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody activates T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells, and/or congenital lymphoid cells (ILCs) and/or modulates bone marrow cells, and wherein the cancer is treated.

42. A method of activating T cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the T cells are activated.

43. A method of activating NK cells of a patient, said method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein said NK cells are activated.

44. A method of activating NKT cells in a patient, the method comprising administering the anti-IL 18-BP antibody of any one of the preceding claims, and wherein the NKT cells are activated.

45. A method of modulating bone marrow cells of a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the bone marrow cells are modulated.

46. A method of activating dendritic cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the dendritic cells are activated.

47. A method of activating dendritic cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the MAIT T cells are activated.

48. A method of activating dendritic cells in a patient, the method comprising administering an anti-IL 18-BP antibody according to any one of the preceding claims, and wherein the γδ T cells are activated.

49. A method of activating ILC cells of a patient, the method comprising administering an anti-IL 18-BP antibody of any one of the preceding claims, and wherein the ILC cells are activated.

50. A method of increasing IL-18-mediated immunostimulatory activity in a Tumor Microenvironment (TME) and/or a lymph node, the method comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody increases IL-18-mediated immunostimulatory activity in the TME and/or lymph node.

51. A method of restoring IL-18 activity on a T cell, NK cell, NKT cell, bone marrow cell, dendritic cell, MAIT T cell, γδ T cell, and/or congenital lymphoid cell (ILC), the method comprising administering an anti-IL 18-BP antibody, wherein the anti-IL 18-BP antibody restores activity on a T cell, NK cell, NKT cell, bone marrow cell, dendritic cell, and/or congenital lymphoid cell (ILC).

52. The method of any one of claims 40-51, wherein the anti-IL 18-BP antibody is administered as a stable liquid pharmaceutical formulation.

53. The method of any one of claims 40 to 52, wherein the T cells are cytotoxic T Cells (CTLs).

54. The method of claim 53, wherein the T cells are selected from the group consisting of CD4 ⁺ T cells and CD8 ⁺ T cells.

55. The method of treatment of any one of claims 40-54, wherein tumor growth inhibition in a subject for treatment is increased by at least about 10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、125％、150％、175％、200％、225％、250％、275％、300％、325％、350％、375％、400％、425％、450％、475％、500％、525％、550％、575％、600％、625％、650％、675％、700％、725％、750％、775％、800％、825％、850％、875％、900％、925％、950％、975％ or 1000% as compared to a control group or the patient not treated.

56. The method of treatment of any one of claims 40-54, wherein the subject for treatment exhibits a reduction in tumor growth of at least about 10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、125％、150％、175％、200％、225％、250％、275％、300％、325％、350％、375％、400％、425％、450％、475％、500％、525％、550％、575％、600％、625％、650％、675％、700％、725％、750％、775％、800％、825％、850％、875％、900％、925％、950％、975％ or 1000% compared to a control group or the patient untreated.

57. The method of any one of claims 40 to 56, wherein the NK cells are cd16+ lymphocytes.

58. The method of any one of claims 40 to 56, wherein the NK cells are cd56+ NK cells.

59. The method of any one of claims 40 to 56, wherein the activation is measured as an increase in expression of one or more activation markers.

60. The method of any one of claims 40 to 59, wherein the activation marker is selected from the group consisting of CD107a, CD137, CD69, granzyme, and perforin.

61. The method of any one of claims 40 to 60, wherein the activation is measured as an increase in proliferation of the NK cells.

62. The method of any one of claims 40-61, wherein the activation is measured as an increase in secretion of one or more cytokines.

63. The method of any one of claims 40-62, wherein the one or more cytokines are selected from the group consisting of ifnγ, TNF, GMCSF, MIG (CXCL 9), IP-10 (CXCL 10), and MCP1 (CCL 2).

64. The method of any one of claims 40-63, wherein the activation is measured as an increase in direct killing of target cells.

65. The method of any one of claims 40 to 64, further comprising administering a second antibody.

66. The method of claim 65, wherein the second antibody is an antibody that binds to and/or inhibits a human checkpoint receptor protein.

67. The method of claim 65 or 66, wherein the second antibody is selected from the group consisting of: anti-PVRIG antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-CTLA-4 antibodies, anti-PD-L2 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-CEACAM-1 antibodies, anti-PVR antibodies, anti-LAG 3 antibodies, anti-CD 112 antibodies, anti-CD 96 antibodies, anti-TIM 3 antibodies, anti-BTLA antibodies, anti-ICOS antibodies, anti-OX 40 antibodies or anti-41 BB antibodies, anti-CD 27 antibodies or anti-GITR antibodies.

68. The method of claim 67, wherein the PVRIG antibody is selected from the group consisting of cha.7.518.1.h4 (S241P) and cha.7.538.1.2.h4 (S241P).

69. The method of claim 67, wherein the anti-PVRIG antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CHA.7.518.1.H4 (S241P) (SEQ ID NO: 260) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CHA.7.518.1.H4 (S241P) (SEQ ID NO: 265).

70. The method of claim 67, wherein the anti-PVRIG antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CHA.7.538.1.2.H4 (S241P) (SEQ ID NO: 270) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CHA.7.538.2.H4 (S241P) (SEQ ID NO: 275).

71. The method of claim 67, wherein the anti-PVRIG antibody comprises: i) Including those from CHA.7.518.4 (SEQ ID NO:1453; FIG. 36 AG) the heavy chain variable domains vhCDR, vhCDR2, and vhCDR3 and ii) the heavy chain variable domain comprising a heavy chain variable domain from CHA.7.518.4 (SEQ ID NO:1457; fig. 36 AG) of the vlCDR, vlCDR2, and vlCDR 3.

72. The method of claim 67, wherein the anti-PVRIG antibody is selected from the group consisting of: GSK4381562/SRF816 (GSK/Surface), NTX2R13 (Nectin Therapeutics), anti-PVRIG antibodies as described in WO 2017/04004, anti-PVRIG antibodies as described in WO 2001008879, anti-PVRIG antibodies as described in WO 2018017864, and anti-PVRIG antibodies as described in WO 2118000205.

73. The method of claim 67, wherein said anti-TIGIT antibody is selected from the group consisting of cpa.9.083.h4 (S241P) and cpa.9.086.h4 (S241P).

74. The method of claim 67, wherein the anti-TIGIT antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CPA.9.083.H4 (S241P) (SEQ ID NO: 350) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CPA.9.083.H4 (S241P) (SEQ ID NO: 355).

75. The method of claim 67, wherein the anti-TIGIT antibody comprises: i) Heavy chain variable domains comprising the vhCDR1, vhCDR2, and vhCDR3 from CPA.9.086.H4 (S241P) (SEQ ID NO: 360) and ii) light chain variable domains comprising the vlCDR 1. VlCDR2, and vlCDR3 from CPA.9.086.H4 (S241P) (SEQ ID NO: 365).

76. The method of claim 67, wherein the anti-TIGIT antibody comprises: i) Including those from CHA.9.547.18 (SEQ ID NO:1177; FIG. 34 QQQQ) of the heavy chain variable domains vhCDR1, vhCDR2 and vhCDR3 and ii) of a polypeptide comprising a polypeptide derived from CHA.9.547.18 (SEQ ID NO:1181; fig. 34 QQQQ) of the light chain variable domains vlCDR, vlCDR2 and vlCDR.

77. The method of claim 67, wherein the anti-TIGIT antibody is selected from the group consisting of: EOS-448 (GlaxoSmithKline, iTeos Therapeutics), BMS-986207, duwanalimumab (AB 154, arcus Biosciences, inc.), AB308 (Arcus Bioscience), european-amber Li Shan antibody (aBGB-A1217, beiGene), tiarey Li Youshan antibody (MTIG7192A,RocheGenentech)、BAT6021(Bio-Thera Solutions)、BAT6005(Bio-Thera Solutions)、IBI939(Innovent Biologics,US2021/00040201)、JS006(Junshi Bioscience/COHERUS)、ASP8374(Astellas Pharma Inc)、 vitamin-Bo Li Shan antibody (MK-7684,Merck Sharp&Dohme), M6332 (MERCK KGAA), ai Tili mab (OMP-313M32,Mereo BioPharma)、SEA-TGT(Seagen)y、HB0030(Huabo Biopharma)、AK127(AKESO)、IBI939(Innovent Biologics), and anti-TIGIT antibodies include Genntech antibodies (MTIG 7192A).

78. The method of claim 67, wherein the anti-PD-1 antibody is selected from the group consisting of: nawuzumab @BMS; CHECKMATE 078) and pembrolizumabMerck), TSR-042 (Tesaro), cimipn Li Shan anti (REGN 2810; regeneron Pharmaceuticals, see US 20170174779), BMS-936559, swadazumab (PDR 001, novartis), dermatitid (CT-011; pfizer Inc.), tirelimumab (BGB-A317, beiGene), carilimumab (SHR-1210, incyte and Jiangsu HengRui), SHR-1210 (CTR 20170299 and CTR 20170322), SHR-1210 (CTR 20160175 and CTR 20170090), xindi Li Shan antibodyElily and), terlipressin (JS 001), JS-001 (CTR), IBI308 (CTR), BGB-a317 (CTR), pemetrexed (AK 105, cepalizumab (Arcus), BAT1306 (Bio-), sashan anti (PF-06801591, pfizer), duotouzumab (), palono (Biocad), carbinikon (), jeromycin (), sikang (), barilizumab (), rauv Corp (Incyte Corp), ceriluzumab (Johnson & Johnson), CS-1003 (), IBI-318 (), ivoriximab (), prtelimumab (), QL-1604 (), 10, terpolizumab (), AZD-7789 (Astrazeneca Plc), bragg antibody (), EMB-02 (), ebenimab (), evelizumab (), rumex (), YBL-006 (Y-biolog) and ONO-4685 () LY-3434172 (ELI LILLY AND Co).

79. The method of claim 67, wherein the anti-PD-L1 antibody is selected from the group consisting of: alemtuzumab @MPDL3280A; IMpower110,110; roche/Genentech, avermectinMSB001071 8C; EMD serrono & Pfizer) and dewaruzumab (MEDI 4736; AstraZeneca). And other antibodies being developed, such as lodalimab (LY 3300054, eli Lily), pi Weishan anti (Jounce Therapeutics Inc), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), en Wo Lishan anti (Jiangsu Simcere Pharmaceutical Co Ltd), shu Geli mab (CStone Pharmaceuticals Co Ltd), ke Xili mab (Checkpoint Therapeutics Inc), parker Mi Lishan anti (CytomX Therapeutics Inc)、IBI-318、IBI-322、IBI-323(Innovent Biologics Inc)、INBRX-105(Inhibrx Inc)、KN-046(Alphamab Oncology)、6MW-3211(Mabwell Shanghai Bioscience Co Ltd)、BNT-311(BioNTech SE)、FS-118(F-star Therapeutics Inc)、GNC-038(Systimmune Inc)、GR-1405(Genrix(Shanghai)Biopharmaceutical Co Ltd)、HS-636(Zhejiang Hisun Pharmaceutical Co Ltd)、LP-002(Lepu Biopharma Co Ltd)、PM-1003(Biotheus Inc)、PM-8001(Biotheus Inc)、STIA-1015(ImmuneOncia Therapeutics LLC)、ATG-101(Antengene Corp Ltd)、BJ-005(BJ Bioscience Inc)、CDX-527(Celldex Therapeutics Inc)、GNC-035(Systimmune Inc)、GNC-039(Systimmune Inc)、HLX-20(Shanghai Henlius Biotech Inc)、JS-003(Shanghai Junshi Bioscience Co Ltd)、LY-3434172(Eli Lilly and Co)、MCLA-145(Merus NV)、MSB-2311(Transcenta Holding Ltd)、PF-07257876(Pfizer Inc)、Q-1802(QureBio Ltd)、QL-301(QLSF Biotherapeutics Inc)、QLF-31907(Qilu Pharmaceutical Co Ltd)、RC-98(RemeGen Co Ltd)、TST-005(Transcenta Holding Ltd)、 alemtuzumab (IMpower 133), BMS-936559/MDX-1105 and/or RG-7446/MPDL3280A, and YW243.55.S70.

80. The method of any one of claims 65-79, wherein the anti-IL 18-BP antibody and the second antibody are administered sequentially or simultaneously in any order and in one or more formulations.

81. The method of any one of claims 40-80, wherein the anti-IL 18-BP antibody is for use in combination with an immunostimulatory antibody, cytokine therapy, an immunomodulatory drug, a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, an anti-hormonal agent, a kinase inhibitor, an anti-angiogenic agent, a cardioprotective agent, an immunosuppressant, an agent that promotes blood cell proliferation, an angiogenesis inhibitor, a Protein Tyrosine Kinase (PTK) inhibitor, or other therapeutic agent.

82. The method of any one of claims 40-81, further comprising administering one or more inflammation small activator.

83. The method of claim 82, wherein the inflammatory body activator is a chemotherapeutic agent.

84. The method of claim 82 or 83, wherein the chemotherapeutic agent is selected from the group consisting of: platinum (including platinum chemotherapeutics), paclitaxel (taxol), sorafenib, doxorubicin, sorafenib, 5-FU, gemcitabine, and irinotecan (CPT-11).

85. The method of claim 84, wherein the platinum chemotherapeutic agent is oxaliplatin or cisplatin.

86. The method of claim 82, wherein the inflammatory body activator is a CD39 inhibitor.

87. The method of claim 86, wherein the CD39 inhibitor is an anti-CD 39 antibody.

88. The method of any one of claims 81-87, wherein the anti-IL 18-BP antibody and the immunostimulatory antibody, cytokine therapy, immunomodulatory drug, cytotoxic agent, chemotherapeutic agent, growth inhibitory agent, anti-hormonal agent, kinase inhibitor, anti-angiogenic agent, cardioprotective agent, immunosuppressive agent, agent that promotes blood cell proliferation, angiogenesis inhibitor, protein Tyrosine Kinase (PTK) inhibitor or other therapeutic agent are administered in any order and in one or more formulations sequentially or simultaneously.

89. The method of treatment of any one of claims 40-88, wherein the cancer is selected from the group consisting of: vascularized tumors, melanomas, non-melanoma skin cancers (squamous cell carcinoma and basal cell carcinoma), mesotheliomas, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine lung cancer (including pleural mesothelioma, neuroendocrine lung cancer), NSCL (large cell), NSCLC large cell adenocarcinoma, non-small cell lung cancer (NSCLC), NSCLC squamous cell carcinoma, soft tissue sarcoma, kaposi's sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, NSCLC with PDL1> =50% tps, Neuroendocrine lung cancer, atypical carcinoid lung cancer, peritoneal cancer, esophageal cancer, hepatocellular cancer, liver cancer (including HCC), stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, urothelial cancer, bladder cancer, liver cancer, glioma, brain cancer (and edema, such as brain tumor-associated edema), breast cancer (including, for example, triple negative breast cancer), testicular cancer, testicular germ cell tumor, colon cancer, colorectal cancer (CRC), colorectal cancer MSS (MSS-CRC); Refractory MSS colorectal cancer; MSS (microsatellite steady state), primary peritoneal carcinoma, primary peritoneal ovarian carcinoma, microsatellite stabilized primary peritoneal carcinoma, platinum resistant microsatellite stabilized primary peritoneal carcinoma, CRC (MSS unknown), rectal cancer, endometrial carcinoma (including endometrial carcinoma), uterine carcinoma, salivary gland carcinoma, renal Cell Carcinoma (RCC), gastroesophageal junction carcinoma, prostate carcinoma, vulval carcinoma, thyroid carcinoma, liver carcinoma, carcinoid, head and neck carcinoma, B cell lymphoma (including non-Hodgkin's lymphoma), low-grade/follicular non-Hodgkin's lymphoma (NHL), small Lymphocyte (SL) NHL, Intermediate/follicular NHL, intermediate diffuse NHL, diffuse large B-cell lymphoma, hyperimmune blast NHL, highly small non-nucleated cell NHL, megaly tumor NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldensted land giant globulinemia, hodgkin's lymphoma (HD), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute Myelogenous Leukemia (AML), hairy cell leukemia, chronic myeloblastic leukemia, Multiple myeloma, post-transplant lymphoproliferative disease (PTLD), abnormal vascular proliferation associated with mole-related hamartoma, meugus syndrome, meckel cell carcinoma, high MSI carcinoma, KRAS mutant tumor, adult T-cell leukemia/lymphoma, adenoid cystic carcinoma (including adenoid cystic carcinoma), melanoma, malignant melanoma, metastatic melanoma, pancreatic carcinoma, ovarian carcinoma (including ovarian carcinoma), pleural mesothelioma, cervical squamous cell carcinoma (cervical SCC), anal squamous cell carcinoma (anal SCC), unidentified primary carcinoma, gallbladder carcinoma, pleural mesothelioma, chordoma, endometrial sarcoma, Chondrosarcoma, uterine sarcoma, uveal melanoma, amyloidosis, AL-amyloidosis, astrocytoma, and myelodysplastic syndrome (MDS).

90. The method of treatment of any one of claims 40-89, wherein the cancer is selected from the group consisting of: clear cell carcinoma (RCC), lung cancer, NSCLC, lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple Negative Breast Cancer (TNBC), head and neck tumors, colorectal adenocarcinoma, melanoma, and metastatic melanoma.

91. The anti-IL 18BP antibody of any one of the preceding claims for use in the treatment of cancer by activating T cells, NK cells, NKT cells, dendritic cells, MAIT T cells, γδ T cells and/or congenital lymphoid cells (ILCs) and/or modulating bone marrow cells of a patient.

92. The anti-IL 18BP antibody of any one of the preceding claims for use in increasing IL-18 mediated immunostimulatory activity in the Tumor Microenvironment (TME) and/or lymph nodes.

93. Use of an anti-IL 18-BP antibody according to any one of the preceding claims for the treatment of cancer in a recipient patient.

94. The anti-IL 18-BP antibody of any one of the preceding claims for use according to any one of the preceding claims.

95. The anti-IL 18-BP antibody of any one of the preceding claims, wherein the anti-IL 18-BP antibody is for use in combination with a second antibody.

96. The anti-IL 18-BP antibody of claim 95, wherein the second antibody is selected from the group consisting of an anti-PVRIG antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-TIGIT antibody.

97. The anti-IL 18-BP antibody of any one of the preceding claims, wherein the anti-IL 18-BP antibody exhibits a binding affinity or KD of less than 0.005pM、0.01pM、0.02pM、0.03pM、0.04pM、0.05pM、0.06pM、0.07pM、0.08pM、0.09pM、0.10pM、0.15pM、0.20pM、0.25pM、0.30pM、0.35pM、0.40pM、0.45pM、0.50pM、0.55pM、0.60pM、0.65pM、0.70pM、0.75pM、0.80pM、0.85pM、0.90pM、0.95pM or 1 pM.