Combination therapy for cancer including CTLA-4 and PD-1 blockers

Abstract

A combination therapy for cancer comprising a CTLA-4 blocker and a PD-1 blocker is disclosed. Specifically, the combination therapy is disclosed in which the CTLA-4 blocker is an effector-silencing anti-CTLA-4 antibody or an effector-silencing anti-CTLA-4 antibody fragment and the PD-1 blocker is an anti-PD-1 or anti-PD-L1 antibody, or an antibody fragment thereof.

Description

Background of the Invention

[0001] (1) Field of Invention

[0002] This invention relates to combination anticancer therapy comprising a CTLA-4 blocker and a PD-1 blocker. Specifically, this invention relates to combination therapy in which the CTLA-4 blocker is an anti-CTLA-4 antibody with reduced or unmeasurable effector function or an anti-CTLA-4 antibody fragment lacking an Fc domain, and the PD-1 blocker is an anti-PD-1 antibody, an anti-PD-1 antibody fragment, an anti-PD-L1 antibody, or an anti-PD-L1 antibody fragment.

[0003] (2) Description of related technologies

[0004] Cancer immunotherapy has taken on a more significant role in treating a variety of cancer indications. Clinical successes in blocking immune-limiting receptors expressed on T cells, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have spurred significant advances in cancer immunotherapy. Blocking CTLA-4 or PD-1 with monoclonal antibodies (mAbs) as a single therapy has led to enhanced antitumor responses and beneficial clinical outcomes in controlled randomized clinical trials.

[0005] A key feature of immune-limiting site blocking for the treatment of various cancers is the clinically validated benefit of combination therapy involving anti-PD-1 and anti-CTLA-4 antibodies. As more clinical data are released, it becomes increasingly clear that anti-PD-1 / CTLA-4 combination therapy may offer superior clinical efficacy compared to targeting either limiting site alone. However, immune-related adverse events (irAEs) associated with anti-CTLA-4 antibodies are significant in both monotherapy settings and in combination therapy with anti-PD-1 antibodies. For example, ipilimumab, the only anti-CTLA-4 antibody marketed by Bristol-Myers Squibb under the brand name YERVOY and approved by the US Food and Drug Administration (FDA), has received a black box warning due to its induction of serious and fatal immune-mediated adverse reactions such as inflammation of the intestines, liver, skin, hormone-producing glands, and / or eyes. Ipilimumab has also been approved for use in combination with the anti-PD-1 antibody nivolumab (marketed by Bristol-Myers Squibb under the brand name Opdivo) for the treatment of advanced renal cell carcinoma and certain colorectal cancers, but due to the significant risk of irAEs, ipilimumab is administered at a low dose or subtherapeutic dose of 1 mg / kg. The subtherapeutic dose for combination therapy is significantly lower than the 3 mg / kg monotherapy dose for unresectable or metastatic melanoma or the 10 mg / kg monotherapy dose for adjuvant melanoma (see YERVOY's Package Insert and Label (July 2018)).

[0006] The FDA-approved anti-PD1 mAbs nivolumab and pembrolizumab are both humanized anti-PD1 IgG4κ antibodies, disclosed in U.S. Patent Nos. 8,008,449 and 8,354,509, respectively. The IgG4 isotype Fc domain is generally considered to have little detectable effector function.

[0007] Ipilimumab is a human anti-CTLA-4 IgG1κ antibody, disclosed in U.S. Patent No. 6,984,720. The heavy chain (HC) constant domain of the IgG1 isotype contains an Fc domain generally considered to have high affinity for the Fc receptor (FcR), which provides significant effector functions to the antibody (e.g., induction of antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC)). Studies have shown that the efficacy of anti-CTLA-4 antibodies requires Fc effector function. For example, Ingram et al., Proc. Natl. Acad. Sci. USA 115: 3912-3917 (2018) showed in a mouse model that the anti-CTLA-4 alpaca heavy chain antibody fragment (V) lacking the heavy chain Fc domain and its associated effector function... HH It has no antitumor activity; however, the antitumor activity of the molecule can be restored by fusing it with the Fc domain of the mouse IgG2 heavy chain, which exhibits effector function; and Selby et al., Cancer Immunol. Res. 1: 32-42 (2013) showed in a mouse model that anti-CTLA-4 antibodies fused to or linked with an Fc domain mutated to eliminate effector function did not show any antitumor activity. See also Simpson et al., J. Exp. Med.;210:1695-710 (2013) and International Patent Application No. WO2014089113.

[0008] Tremelizumab is a human anti-CTLA-4 human IgG2 antibody, disclosed in U.S. Patent No. 8,491,895. Human IgG2 isotypes have been selected to minimize potential effector functional activity and thus potentially reduce irAEs. However, as Vargas et al., Cancer Cell 33: 649-663 (2018), tremelizumab retains effector function; and Bertrand et al., BMC Med. 13: 211-214 (2015) showed that although tremelizumab can be administered at higher doses than ipilimumab, it can still induce irAEs, particularly intestinal and skin inflammatory immune-mediated toxicities. See also Ribas et al., The Oncologist 12: 873-993 (2007), Schneider-Merck et al., J. Immunol. 184: 512-520 (2010), and Könitzer et al., PLoS One. 10:e0145633 (2015), which showed other IgG2 antibodies that induce ADCC and ADCP in vitro with similar equivalence to human IgG1 isotypes.

[0009] Other attempts to reduce ipilimumab irAEs include BMS-986249, a probody consisting of ipilimumab linked to a patented masking peptide covering the active antigen binding site of the antibody via a protease-cleavable linker. The masking peptide can reduce irAEs by minimizing the ability of ipilimumab to bind to CTLA-4 in normal tissues (see International Patent Applications WO2009025846, WO2010081173, WO2018222949, WO2018085555, Pai et al., J. Clin. Invest. 129: 349-363 (2019) and Korman et al., Abstract SY09-01, AACR Annual Meeting Vol. 77, No. 13 (2017)).

[0010] Based on the indications, the therapeutic efficacy of anti-CTLA-4 antibodies such as ipilimumab may involve the regulation of T cells (T cells). regs Studies excluding certain antibodies have suggested that anti-CTLA-4 antibodies with enhanced ADCC activity, such as ipilimumab, would provide more potent antitumor activity than current antibodies. U.S. Patent No. 10,196,445 discloses several ipilimumab variants with enhanced ADCC activity.

[0011] Some standard-of-care treatments for cancer include providing anti-PD-1 antibodies in combination with chemotherapy. The anti-tumor activity of anti-CTLA-4 antibodies can further enhance the efficacy of these therapies; however, because gastrointestinal toxicity is one of the most common side effects experienced during chemotherapy, adding anti-CTLA-4 antibodies to the therapy may actually exacerbate gastrointestinal toxicity.

[0012] Clearly, enabling the administration of anti-CTLA-4 antibodies at higher, more optimal levels without associated irAEs, particularly the skin and gut inflammatory immune-mediated toxicities associated with current anti-CTLA-4 antibodies, would likely allow for more effective therapies in combination with anti-PD-1 antagonists and, optionally, chemotherapy. Invention Overview

[0014] The inventors have discovered that while certain CTLA-4 blockers, when administered as monotherapy, exhibit reduced or no measurable antitumor activity when combined with CTLA-4, they may demonstrate clinically relevant antitumor activity when used in combination therapy with PD-1 blockers. The inventors have also discovered that these certain CTLA-4 blockers can exert antitumor activity in CTLA-4 / PD-1 blocker combination therapy without inducing immune-mediated adverse reactions (irAEs), including irAEs of the skin and intestines associated with currently approved CTLA-4 / PD-1 blocker combination therapies. The CTLA-4 / PD-1 blocker combinations disclosed herein enable therapies with an increased therapeutic index compared to current CTLA-4 / PD-1 blocker combination therapies (including combination therapies including chemotherapy), which could lead to more effective cancer treatment with improved patient outcomes.

[0015] Certain CTLA-4 blockers used as part of the CTLA-4 / PD-1 blocking combination therapy of the present invention may be selected from (i) effector-silencing anti-CTLA-4 antibodies and (ii) effector-silencing anti-CTLA-4 antibody fragments that lack a crystallizable fragment (Fc) domain or have a missing Fc domain containing those regions of the Fc domain that bind Fc receptors (FcRs). The effector-silencing antibody or antibody fragment exhibits (i) no measurable binding to one or more FcRs, as measurable in a Biacore assay, wherein an association constant in the micromolar range indicates no measurable binding or (ii) reduced measurable binding to one or more FcRs compared to typical binding to the same isotype antibody, as measurable in a Biacore assay. These certain CTLA-4 blockers are effector-silencing CTLA-4 blockers.

[0016] In certain implementations, these effector-silencing anti-CTLA-4 antibodies and effector-silencing anti-CTLA-4 antibody fragments may not exhibit measurable antitumor activity in anticancer monotherapy, but will exhibit measurable antitumor activity in combination anticancer therapy with PD-1 or PD-L1 blockers, and will not exhibit irAEs generally associated with CTLA-4 / PD-1 blocker combination therapy, particularly skin or intestinal inflammatory immune-related toxicities.

[0017] The effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment disclosed herein can be used in combination with PD-1 or PD-L1 blockers at higher doses and for longer periods without exhibiting irAEs generally associated with CTLA-4 / PD-1 blocking combination therapy, particularly skin or intestinal inflammatory immune-related toxicities. Currently, the approved dose of the anti-CTLA-4 antibody ipilimumab for anti-CTLA-4 / PD-1 blocking combination therapy is 1 mg / kg compared to the approved dose of 3 mg / kg or 10 mg / kg for monotherapy (see YERVOY's packaging insert and label (July 2018)) or the fixed dose of 100 mg or less anticipated for CTLA-4 / PD-1 blocking combination therapy as described in International Patent Application WO2018183408. Therefore, the CTLA-4 / PD-1 blocking combination therapy of the present invention can use effector-silencing anti-CTLA-4 antibodies or effector-silencing anti-CTLA-4 antibody fragments at doses that are currently approved for use in monotherapy or at higher doses than those currently approved for use in anti-CTLA-4 antibodies. The effector-silencing anti-CTLA-4 antibodies or anti-CTLA-4 antibody fragments disclosed herein can also be used in combination with anti-PD-1 or anti-PD-L1 antibodies at doses similar to those currently used or intended for use in CTLA-4 / PD-1 blocking combination therapy, but achieving a longer duration of action than currently achievable with anti-CTLA-4 antibodies, and without exhibiting the irAEs generally associated with CTLA-4 / PD-1 blocking combination therapy, particularly skin or intestinal inflammatory immune-related toxicities.

[0018] Therefore, the present invention provides a combination therapy for treating cancer in individuals requiring such treatment, the method comprising administering to an individual with cancer (i) a therapeutic dose of a PD-1 or PD-L1 blocker and (ii) a therapeutic dose of an effector-silencing CTLA-4 blocker to treat the cancer, wherein the effector-silencing CTLA-4 blocker exhibits antitumor activity in the combination therapy that would not be exhibited when administered to the individual in a monotherapy without a PD-1 or PD-L1 blocker. In a further embodiment, compared to combination therapy comprising an anti-CTLA-4 antibody exhibiting effector function, the combination therapy does not induce or has a reduced risk of inducing immune-mediated adverse events (irAEs) in the gut or skin during the course of the combination therapy, said adverse events being greater than grade 2 as defined in the Common Terminology Criteria for Adverse events (CTCAE) version 5.0. In a particular embodiment, the effector silencing CTLA-4 blocker used as part of the combination therapy does not induce grade 2 or higher irAEs in the skin or intestine for at least the first 10 weeks of the combination therapy. In a particular embodiment, the combination therapy does not cause detectable irAEs for at least the first four weeks of the combination therapy, or does not cause grade 1 or higher irAEs for at least the first four weeks of the combination therapy.

[0019] In one implementation, the effector silencing CTLA-4 blocker used as part of the combination therapy described herein is an effector silencing anti-CTLA-4 antibody or an effector silencing anti-CTLA-4 antibody fragment.

[0020] In another embodiment, the PD-1 blocker used as part of the combination therapy described herein is an anti-PD-1 or anti-PD-L1 antibody or an anti-PD-1 or anti-PD-L1 antibody fragment. In a particular embodiment, the anti-PD-1 or anti-PD-L1 antibody comprises an HC domain containing one or more mutations in the Fc domain that silence the antibody effector. The PD-1 blocker may also be an anti-PD-1 or anti-PD-L1 antibody fragment, each of which lacks an Fc domain or those regions of the Fc domain that bind one or more FcRs, thereby silencing the antibody fragment effector.

[0021] The present invention further provides a combination therapy for cancer, comprising administering to an individual in need of cancer therapy (i) a first formulation comprising a PD-1 blocking agent selected from an anti-PD-1 antibody having an IgG4 or IgG2 Fc domain, an effector-silencing anti-PD-1 antibody, and an effector-silencing anti-PD-1 antibody fragment; and (ii) a second formulation comprising an effector-silencing CTLA-4 blocking agent selected from an effector-silencing anti-CTLA-4 antibody and an effector-silencing anti-CTLA-4 antibody fragment.

[0022] The present invention further provides a combination therapy for cancer, comprising administering to an individual in need of cancer therapy a formulation comprising (i) a PD-1 blocking agent selected from an anti-PD-1 antibody having an IgG4 or IgG2 Fc domain, an effector-silencing anti-PD-1 antibody, and an effector-silencing anti-PD-1 antibody fragment; and (ii) an effector-silencing CTLA-4 blocking agent selected from an effector-silencing anti-CTLA-4 antibody and an effector-silencing anti-CTLA-4 antibody fragment.

[0023] The present invention further provides a combination therapy for cancer, comprising administering to an individual in need of cancer therapy (i) a first formulation comprising a PD-L1 blocking agent selected from an anti-PD-L1 antibody having an IgG4 or IgG2 Fc domain, an effector-silencing anti-PD-L1 antibody, and an effector-silencing anti-PD-L1 antibody fragment; and (ii) a second formulation comprising an effector-silencing CTLA-4 blocking agent selected from an effector-silencing anti-CTLA-4 antibody and an effector-silencing anti-CTLA-4 antibody fragment.

[0024] The present invention further provides a combination therapy for cancer treatment comprising administering to an individual in need of cancer therapy a formulation comprising (i) a PD-L1 blocking agent selected from an anti-PD-L1 antibody having an IgG4 or IgG2 Fc domain, an effector-silencing anti-PD-L1 antibody, and an effector-silencing anti-PD-L1 antibody fragment; and (ii) an effector-silencing CTLA-4 blocking agent selected from an effector-silencing anti-CTLA-4 antibody and an effector-silencing anti-CTLA-4 antibody fragment.

[0025] In a more specific implementation of the combination therapy, the effector-silencing anti-CTLA-4 antibody contains an IgG1 Fc domain, which has (i) starting from amino acid position 297 N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylated, or further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions of the Fc domain; (ii) amino acid substitution mutations selected from N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, N297A / D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A / N297G, or further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions of the Fc domain; Or a mutant Fc domain with 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A, or further containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0026] In a specific implementation of the combination therapy, the effector-silencing anti-CTLA-4 antibody contains an IgG2 Fc domain, which has (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylated, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from N297A / D265S, D265A, P329G / D265A / N297G or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0027] In a specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution, and further comprises (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - A glycosylated Fc domain, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N267A, P329G, and D265A / N297A, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylation, and amino acid substitution mutations selected from N267A, P329G and D265A / N297A, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0028] In a further implementation of the combination therapy, the effector silencing anti-CTLA-4 antibody fragment lacking the Fc domain is or contains a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), or an antigen-binding fragment dimer F(ab')2.

[0029] In a specific implementation of the combination therapy, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment comprises three heavy chain (HC) complementarity-determining regions (CDRs) and three light chain (LC) CDRs of an anti-CTLA-4 antibody selected from ipilimumab, tremelimumab, REGN4659, AGEN1884w, 8D2 / 8D2 (RE), 8D2 / 8D2 (RE)-variant 1, 8D2H1L1, 8D2H1L1-variant 1, 8D2H2L2, 8D2H2L2-variant 1, 8D3H3L3, 8D2H2L15, 8D2H2L15-variant 1, 8D2H2L17, and 8D2H2L17-variant 1.

[0030] In certain implementations of combination therapy, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment contains ipilimumab's V... H and V L Trimelimumab V H and V L REGN4659's V H and V L ,AGEN1884w's V H and V L V of 8D2 / 8D2 (RE) H and V L V of 8D2 / 8D2 (RE)-variant 1 H and V L 8D2H1L1 of V H and V L V of 8D2H1L1-variant 1 H and V L V of 8D2H2L2 H and V L V of 8D2H2L2- variant 1 H and V L8D3H3L3 of V H and V L 8D2H2L15 of V H and V L V of 8D2H2L15-variant 1 H and V L V of 8D2H2L17 H and V L Or V of 8D2H2L17-variant 1 H and V L .

[0031] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment comprises (i) a V containing the amino acid sequence shown in SEQ ID NO:7. H and V containing the amino acid sequence shown in SEQ ID NO:8 L (ii) V containing the amino acid sequence shown in SEQ ID NO:15 H and V containing the amino acid sequence shown in SEQ ID NO:16 L (iii) V containing the amino acid sequence shown in SEQ ID NO:95 H and V containing the amino acid sequence shown in SEQ ID NO:96 L ; or (iv) V having the amino acid sequence shown in SEQ ID NO:97 H and V having the amino acid sequence shown in SEQ ID NO:98 L .

[0032] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment comprises (i) a V containing the amino acid sequence shown in SEQ ID NO:73. H The domain and V containing the amino acid sequence shown in SEQ ID NO:74 L (ii) a VH domain containing the amino acid sequence shown in SEQ ID NO:75 and a V domain containing the amino acid sequence shown in SEQ ID NO:76. L (iii) a domain containing the amino acid sequence shown in SEQ ID NO:77. H The domain and V containing the amino acid sequence shown in SEQ ID NO:78 L (iv) A V domain comprising the amino acid sequence shown in SEQ ID NO: 79 H The domain and V containing the amino acid sequence shown in SEQ ID NO:80 LDomain; (v) V containing the amino acid sequence shown in SEQ ID NO:81 H The domain and V containing the amino acid sequence shown in SEQ ID NO:82 L Domain; (vi) V containing the amino acid sequence shown in SEQ ID NO:83 H The domain and V containing the amino acid sequence shown in SEQ ID NO:84 L Domain; (vii) V containing the amino acid sequence shown in SEQ ID NO:85 H The domain and V containing the amino acid sequence shown in SEQ ID NO:86 L (viii) A domain containing the amino acid sequence shown in SEQ ID NO:87. H The domain and V containing the amino acid sequence shown in SEQ ID NO:88 L Domain; (ix) V containing the amino acid sequence shown in SEQ ID NO:89 H The domain and V containing the amino acid sequence shown in SEQ ID NO:90 L Domain; (x) V containing the amino acid sequence shown in SEQ ID NO:91 H The domain and V containing the amino acid sequence shown in SEQ ID NO:92 L Domain; or (xi) containing the amino acid sequence shown in SEQ ID NO:93. H The domain and V containing the amino acid sequence shown in SEQ ID NO:94 L Structural domain.

[0033] In a further embodiment of the combination therapy, the effector silencing CTLA-4 blocker is an effector silencing anti-CTLA-4 antibody selected from the effector silencing anti-CTLA-4 antibodies disclosed in Table 4-18.

[0034] In a further embodiment of the combination therapy, the effector-silencing CTLA-4 binder is an effector-silencing anti-CTLA-4 antibody fragment containing one or more immunoglobulin single variable domains (ISVDs), each ISVD containing a variable domain (V) of a camel-only heavy chain antibody. HHHowever, none of the ISVDs described herein contains a CDR1 containing the amino sequence FYGMG (SEQ ID NO: 69), a CDR2 containing the amino acid sequence DIRTSAGRTTYADSVKG (SEQ ID NO: 70), or a CDR3 containing the amino acid EMSGISGWDY (SEQ ID NO: 71) or EPSGISGWDY (SEQ ID NO: 72), as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087588. HH or V containing one, two, or three mutations in CDR3 as disclosed in WO2008071447. HH However, ISVDs containing the CDRs are not excluded from the conditions only in embodiments in which one or more ISVDs are fused or linked to an effector silencing heterologous HC domain or Fc domain, including, for example, any effector silencing antibody HC domain or Fc domain disclosed herein.

[0035] In a specific embodiment of the combination therapy, the anti-PD-1 antibody or anti-PD-1 antibody fragment comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs of pembrolizumab, nivolumab, or cemiplimab-rwlc. In a specific embodiment of the combination therapy, the anti-PD-1 antibody comprises (i) the V of pembrolizumab. H and V L (ii) V of nivolumab H and V L ; or (iii) cemiplimab-rwlc of V H and V L .

[0036] In a further embodiment, the anti-PD-1 antibody or anti-PD-1 antibody fragment comprises (i) a V having the amino acid sequence shown in SEQ ID NO:29. H and V having the amino acid sequence shown in SEQ ID NO:30 L (ii) V having the amino acid sequence shown in SEQ ID NO:23 H and V having the amino acid sequence shown in SEQ ID NO:24 L ; or (iii) V having the amino acid sequence shown in SEQ ID NO:99 H and V having the amino acid sequence shown in SEQ ID NO:100 LIn a further embodiment, the anti-PD1 antibody comprises (i) an HC having the amino acid sequence shown in SEQ ID NO:27 and an LC having the amino acid sequence shown in SEQ ID NO:28; (ii) an HC having the amino acid sequence shown in SEQ ID NO:25 and an LC having the amino acid sequence shown in SEQ ID NO:26; or (iii) an HC having the amino acid sequence shown in SEQ ID NO:101 and an LC having the amino acid sequence shown in SEQ ID NO:102.

[0037] In a specific implementation of the combination therapy, the anti-PD-L1 antibody or anti-PD-L1 antibody fragment comprises (i) the V of atezolizumab. H and V L (ii) V of avelumab H and V L Domain; or, (iii) the V of durvalumab H and V L Structural domain.

[0038] In a further embodiment; the anti-PD-L1 antibody or anti-PD-L1 antibody fragment comprises (i) a V containing the amino acid sequence shown in SEQ ID NO: 103. H The domain and V containing the amino acid sequence shown in SEQ ID NO:104 L (ii) a domain containing the amino acid sequence shown in SEQ ID NO:105. H The domain and V containing the amino acid sequence shown in SEQ ID NO:106 L (iii) a domain; or (iii) a V containing the amino acid sequence shown in SEQ ID NO:107. H The domain and V containing the amino acid sequence shown in SEQ ID NO:108 L Structural domain.

[0039] In a specific implementation of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody may contain an IgG1, IgG2, or IgG4 Fc domain as disclosed herein, which may contain a C-terminal lysine or be lacking a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0040] In a specific embodiment of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody comprises (i) an IgG2 or IgG4 Fc domain; and (ii) an IgG1, IgG2, or IgG4 Fc domain comprising a domain containing an amino acid starting at position 297.N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylated, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions; (iii) IgG1 Fc domains comprising N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, L235G / G236R, S267E / L328F, S2339D / A330L / I332E or D265A / N297G amino acid substitutions, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions; (iv) IgG2 Fc domains comprising amino acid substitutions of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (v)IgG4 Fc domains comprising amino acid substitutions of S228P and amino acid substitutions of N267A, P329G, or D265A / N297A, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions are identified according to Eu numbering.

[0041] In a specific implementation of the combination therapy, the PD-1 blocker is an anti-PD-1 antibody selected from the anti-PD-1 antibodies disclosed in Tables 19-27 or an anti-PD-L1 antibody selected from the anti-PD-L1 antibodies disclosed in Tables 28-36.

[0042] In a further implementation of the combination therapy, the anti-PD-1 antibody fragment or anti-PD-L1 antibody fragment lacking the Fc domain is a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), or an antigen-binding fragment dimer F(ab')2.

[0043] In a further embodiment of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody fragment comprises one or more ISVDs, each ISVD containing a V of camel-only heavy chain antibody. HH .

[0044] In certain implementations of combination therapy, the CTLA-4 blocker is administered at a dose containing approximately 1 mg / kg to approximately 3 mg / kg of CTLA-4 blocker or at a fixed dose of more than approximately 100 mg of CTLA-4 blocker, regardless of individual weight.

[0045] In certain implementations of the combination therapy, the CTLA-4 blocker is administered at a dose containing 1 mg / kg to 3 mg / kg of CTLA-4 blocker.

[0046] In certain implementations of the combination therapy, the CTLA-4 blocker is administered at a dose containing 3 mg / kg to 10 mg / kg of CTLA-4 blocker.

[0047] In certain implementations of the combination therapy, the CTLA-4 blocker is administered at a dose containing more than about 10 mg / kg of CTLA-4 blocker.

[0048] In certain implementations of combination therapy, the PD-1 blocker is administered at a dose of about 2 or 3 mg / kg or more, or at a fixed dose of about 200 mg or more, regardless of the individual’s weight.

[0049] In certain implementations of combination therapy, the PD-1 blocker is administered at a dose ranging from 200 mg to 400 mg by weight, regardless of the individual.

[0050] In a specific implementation of the combination therapy, the PD-1 blocker is administered at a dose of 400 mg, independent of individual weight.

[0051] In certain implementations of the combination therapy, a PD-1 blocker is administered to the individual first, followed by a CTLA-4 blocker, or a CTLA-4 blocker is administered to the individual first, followed by a PD-1 blocker. In certain implementations, the PD-1 blocker and CTLA-4 blocker are administered simultaneously.

[0052] In a specific implementation of combination therapy, an individual is administered a chemotherapeutic agent before, concurrently with, or after combination therapy. In a specific implementation, the chemotherapeutic agent is selected from actinomycin, all-trans retinoic acid, alitretinoin, azathioprine, azathioprine, bexarotene, bleomycin, bortezomib, carmoflurane, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doceflurane, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib (IM) Atinib, ixabepilone, irinotecan, nitrogen mustard, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, nitrofluridine, temozolomide (oral dacarbazine), teniposide, thioguanine, topotecan, utidelone, penoxuridine, vemurafenib, vincristine, vinblastine, vindesine, vincaenoline, and vorinostat.

[0053] In a specific implementation plan for combination therapy, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, kidney cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0054] In a specific implementation plan for combination therapy, the cancer is pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

[0055] In a specific implementation of the combination therapy, the individual is a human, the CTLA-4 blocker is combined with human CTLA-4, the PD-1 blocker is combined with human PD-1, and the PD-L1 blocker is combined with human PD-L1.

[0056] Antibodies and Compositions

[0057] This invention further provides an effector-silencing anti-CTLA-4 antibody or an effector-silencing anti-CTLA-4 antibody fragment, each containing V H and V LV H Contains three heavy chain CDRs and V L It contains three light chain CDRs that bind together to CTLA-4. In a particular embodiment, CTLA-4 is human CTLA-4.

[0058] In a more specific implementation, the effector-silencing anti-CTLA-4 antibody comprises an IgG1 Fc domain, which has (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylated, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, but effector silencing is required. Anti-CTLA-4 antibodies do not include ipilimumab consisting only of N297A substitutions; (ii) selected from N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D Amino acid substitution mutations of 265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, N297A / D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A / N297G, or further containing mutations of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain; or (iii) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A, or further containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0059] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody comprises an IgG2 Fc domain, which has (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylated, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from N297A / D265S, D265A, P329G / D265A / N297G or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0060] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution, and further comprises (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - A glycosylated Fc domain, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N267A, P329G, and D265A / N297A, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N- Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from N267A, P329G and D265A / N297A, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0061] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment comprises three heavy chain (HC) complementarity-determining regions (CDRs) and three light chain (LC) CDRs of an anti-CTLA-4 antibody selected from ipilimumab, trimemumab, REGN4659, AGEN1884w, 8D2 / 8D2 (RE), 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15, and 8D2H2L17.

[0062] In a particular implementation, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment contains the V of ipilimumab. H and V L Trimelimumab V H and V L REGN4659's V H and V L ,AGEN1884w's V H and V L V of 8D2 / 8D2 (RE) H and V L 8D2H1L1 of V H and V L V of 8D2H2L2 H and V L 8D3H3L3 of V H and V L 8D2H2L15 of V H and V L Or V of 8D2H2L17 H and V L .

[0063] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment contains the V of 8D2 / 8D2(RE)-variant 1. H and V L V of 8D2H1L1-variant 1 H and VL V of 8D2H2L2- variant 1 H and V L V of 8D2H2L15-variant 1 H and V L Or V of 8D2H2L17-variant 1 H and V L These variants contain V H The substitution of methionine by isoleucine at position 18 in the amino acid sequence.

[0064] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the anti-effector-silencing CTLA-4 antibody fragment comprises (i) a V having the amino acid sequence shown in SEQ ID NO:7. H and V having the amino acid sequence shown in SEQ ID NO:8 L (ii) V having the amino acid sequence shown in SEQ ID NO:15 H and V having the amino acid sequence shown in SEQ ID NO:16 L (iii) V having the amino acid sequence shown in SEQ ID NO:95 H and V having the amino acid sequence shown in SEQ ID NO:96 L ; or (iv) V having the amino acid sequence shown in SEQ ID NO:97 H and V having the amino acid sequence shown in SEQ ID NO:98 L .

[0065] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody or the effector-silencing anti-CTLA-4 antibody fragment comprises (i) a V containing the amino acid sequence shown in SEQ ID NO:73. H The domain and V containing the amino acid sequence shown in SEQ ID NO:74 L (ii) a VH domain containing the amino acid sequence shown in SEQ ID NO:75 and a V domain containing the amino acid sequence shown in SEQ ID NO:76. L (iii) a domain containing the amino acid sequence shown in SEQ ID NO:77. H The domain and V containing the amino acid sequence shown in SEQ ID NO:78 L (iv) A V domain comprising the amino acid sequence shown in SEQ ID NO: 79 H The domain and V containing the amino acid sequence shown in SEQ ID NO:80 LDomain; (v) V containing the amino acid sequence shown in SEQ ID NO:81 H The domain and V containing the amino acid sequence shown in SEQ ID NO:82 L Domain; (vi) V containing the amino acid sequence shown in SEQ ID NO:83 H The domain and V containing the amino acid sequence shown in SEQ ID NO:84 L Domain; (vii) V containing the amino acid sequence shown in SEQ ID NO:85 H The domain and V containing the amino acid sequence shown in SEQ ID NO:86 L (viii) A domain containing the amino acid sequence shown in SEQ ID NO:87. H The domain and V containing the amino acid sequence shown in SEQ ID NO:88 L Domain; (ix) V containing the amino acid sequence shown in SEQ ID NO:89 H The domain and V containing the amino acid sequence shown in SEQ ID NO:90 L Domain; (x) V containing the amino acid sequence shown in SEQ ID NO:91 H The domain and V containing the amino acid sequence shown in SEQ ID NO:92 L Domain; or (xi) containing the amino acid sequence shown in SEQ ID NO:93. H The domain and V containing the amino acid sequence shown in SEQ ID NO:94 L Structural domain.

[0066] In a particular implementation, the effector silencing anti-CTLA-4 antibody fragment is selected from F(ab), F(ab')2, Fv, and scFv.

[0067] In a further embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD containing the variable domain of camel-only heavy chain antibodies (V... HHHowever, none of the ISVDs mentioned must contain a CDR1 containing the amino sequence FYGMG (SEQ ID NO: 69), a CDR2 containing the amino acid sequence DIRTSAGRTTYADSVKG (SEQ ID NO: 70), or a CDR3 containing the amino acid EMSGISGWDY (SEQ ID NO: 71) or EPSGISGWDY (SEQ ID NO: 72), as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087588. HH or V containing one, two, or three mutations in CDR3 as disclosed in WO2008071447. HH However, ISVDs containing the CDRs are not excluded from the conditions only in embodiments in which one or more ISVDs are fused or linked to an effector silencing heterologous HC domain or Fc domain, including, for example, any effector silencing antibody HC domain or Fc domain disclosed herein.

[0068] The present invention further provides each of the effector-silencing anti-CTLA-4 antibodies disclosed in Tables 4-18, but the effector-silencing anti-CTLA-4 antibodies do not include ipilimumab composed only of N297A substitution.

[0069] The present invention further provides compositions comprising an effector-silencing anti-CTLA-4 antibody or an effector-silencing anti-CTLA-4 antibody fragment as disclosed herein and a pharmaceutically acceptable carrier.

[0070] The present invention further provides an anti-PD-1 antibody, comprising...

[0071] (a) Possesses HC variable structural domain (V H Heavy chains (HC) and those with LC variable structural domains (V) L The light chain (LC) of ) where (i)V H Contains at least three HC-complementarity-determining regions (CDRs) of pembrolizumab and V L At least three LC-CDRs containing pembrolizumab, (ii) V H At least three HC-CDRs containing nivolumab, and V L At least three LC-CDRs containing nivolumab, or (iii) V H At least three HC-CDRs containing cemiplimab-rwlc, and V L At least three LC-CDRs containing cemiplimab-rwlc, and

[0072] (b) An IgG1, IgG2, or IgG4 Fc domain containing (i) an amino acid starting at position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylated, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) IgG1 Fc domains comprising N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, N297A / D356E / L358M, L234F / L235E / P331S / D356E / L358M, or D265A / N297G amino acid substitutions, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iii) IgG2 Fc domains comprising amino acid substitutions of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iv) IgG4 Fc domains comprising amino acid substitutions of S228P and amino acid substitutions of N267A, P329G, or D265A / N297A, or further comprising mutated Fc domains with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions are identified according to Eu numbering.

[0073] In a further embodiment of the above-described anti-PD-1 antibody, the anti-PD-1 antibody comprises (i) V having the amino acid sequence shown in SEQ ID NO: 29. H and V having the amino acid sequence shown in SEQ ID NO:30 L (ii) V having the amino acid sequence shown in SEQ ID NO:23 H and V having the amino acid sequence shown in SEQ ID NO:24 L ; or (iii) V having the amino acid sequence shown in SEQ ID NO:99 H and V having the amino acid sequence shown in SEQ ID NO:100 L .

[0074] In specific embodiments of the anti-PD-1 antibody, the IgG1, IgG2, or IgG4 Fc domain, as disclosed herein, may further include or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0075] The present invention further provides an anti-PD-1 antibody fragment comprising an HC variable domain (V H Heavy chains (HC) and those with LC variable structural domains (V) L The light chain (LC) of ) where (i)V H Contains at least three HC-complementarity-determining regions (CDRs) of pembrolizumab and V L At least three LC-CDRs containing pembrolizumab, (ii) V H At least three HC-CDRs containing nivolumab, and V L At least three LC-CDRs containing nivolumab, or (iii) V H At least three HC-CDRs containing cemiplimab-rwlc, and V L At least three LC-CDRs containing cemiplimab-rwlc.

[0076] In a further embodiment of the anti-PD-1 antibody fragment, the anti-PD-1 antibody fragment comprises (i) a V having the amino acid sequence shown in SEQ ID NO:29. H and V having the amino acid sequence shown in SEQ ID NO:30 L (ii) V having the amino acid sequence shown in SEQ ID NO:23 H and V having the amino acid sequence shown in SEQ ID NO:24 L ; or (iii) V having the amino acid sequence shown in SEQ ID NO:99 H and V having the amino acid sequence shown in SEQ ID NO:100 L .

[0077] In a specific embodiment of the above-described anti-PD-1 antibody fragment, the anti-PD-1 antibody fragment is selected from F(ab), F(ab')2, Fv, and scFv.

[0078] The present invention further provides each of the anti-PD-1 antibodies disclosed in Tables 19-27.

[0079] The present invention further provides compositions comprising an anti-PD-1 antibody or an anti-PD-1 antibody fragment as disclosed herein and a pharmaceutically acceptable carrier.

[0080] The present invention further provides an anti-PD-L1 antibody, comprising...

[0081] (a) Possesses HC variable structural domain (V H Heavy chains (HC) and those with LC variable structural domains (V) L The light chain (LC) of ) where (i)V H Contains at least three HC-complementarity-determining regions (CDRs) of devalumab and V L At least three LC-CDRs containing devalumab, (ii) V H At least three HC-CDRs containing acimetidine, and V L At least three LC-CDRs containing acimetidine, or (iii) V H At least three HC-CDRs containing atezolizumab, and V L At least three LC-CDRs containing atezolizumab, and

[0082] (b) An IgG1, IgG2, or IgG4 Fc domain containing (i) an amino acid starting at position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylated, or Fc domains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) IgG1 Fc domains containing N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, N297A / D356E / L358M, L234F / L235E / P331S / D356E / L358M, or D265A / N297G amino acid substitutions, or Fc domains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iii) IgG2 Fc domains containing amino acid substitutions of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or Fc domains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iv) IgG4 Fc domains containing S228P amino acid substitutions and N267A, P329G, or D265A / N297A amino acid substitutions, or Fc domains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions are identified according to the Eu number, but only if V H and V L When the amino acid sequences of SEQ ID NO:107 and SEQ ID NO:108 are respectively present, then the heavy chain (HC) constant domain is not an IgG1 isotype with the N297A / D356E / L358M substitution combination or when V H and V L When the amino acid sequences of SEQ ID NO:103 and SEQ ID NO:104 are respectively present, then the HC constant domain is not an IgG1 isotype with the substitution combination of L234F / L235E / P331S / D356E / L358M.

[0083] In a further embodiment of the above-described anti-PD-L1 antibody, the anti-PD-L1 antibody comprises (i) V having the amino acid sequence shown in SEQ ID NO:103. H and V having the amino acid sequence shown in SEQ ID NO:104 L (ii) V having the amino acid sequence shown in SEQ ID NO:105 H and V having the amino acid sequence shown in SEQ ID NO:106 L; or (iii) V having the amino acid sequence shown in SEQ ID NO:107 H and V having the amino acid sequence shown in SEQ ID NO:108 L .

[0084] In specific embodiments of the anti-PD-L1 antibody, the IgG1, IgG2, or IgG4 Fc domain, as disclosed herein, may further include or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0085] The present invention further provides an anti-PD-L1 antibody fragment comprising an HC variable domain (V H Heavy chains (HC) and those with LC variable structural domains (V) L The light chain (LC) of ) where (i)V H Contains at least three HC-complementarity-determining regions (CDRs) of devalumab and V L At least three LC-CDRs containing devalumab, (ii) V H At least three HC-CDRs containing acimetidine, and V L At least three LC-CDRs containing acimetidine, or (iii) V H At least three HC-CDRs containing atezolizumab, and V L At least three LC-CDRs containing atezolizumab.

[0086] In a further embodiment, the anti-PD-L1 antibody fragment comprises (i) a V having the amino acid sequence shown in SEQ ID NO:103. H and V having the amino acid sequence shown in SEQ ID NO:104 L (ii) V having the amino acid sequence shown in SEQ ID NO:105 H and V having the amino acid sequence shown in SEQ ID NO:106 L ; or (iii) V having the amino acid sequence shown in SEQ ID NO:107 H and V having the amino acid sequence shown in SEQ ID NO:108 L .

[0087] In a specific embodiment of the above-described anti-PD-L1 antibody fragment, the anti-PD-1 antibody fragment is selected from F(ab), F(ab')2, Fv, and scFv.

[0088] This invention further provides each of the anti-PD-L1 antibodies disclosed in Tables 28-36, but subject to VH and V L When the amino acid sequences of SEQ ID NO:107 and SEQ ID NO:108 are respectively present, then the heavy chain (HC) constant domain is not an IgG1 isotype with the N297A / D356E / L358M substitution combination or when V H and V L When the amino acid sequences of SEQ ID NO:103 and SEQ ID NO:104 are respectively present, then the HC constant domain is not an IgG1 isotype with the substitution combination of L234F / L235E / P331S / D356E / L358M.

[0089] The present invention further provides compositions comprising an anti-PD-L1 antibody or an anti-PD-L1 antibody fragment disclosed herein and a pharmaceutically acceptable carrier.

[0090] The present invention further provides compositions comprising (i) the anti-CTLA-4 antibody and the anti-PD-1 antibody disclosed herein, and a pharmaceutically acceptable carrier; or (ii) the anti-CTLA-4 antibody and the anti-PD-L1 antibody disclosed herein, and a pharmaceutically acceptable carrier.

[0091] The present invention further provides compositions comprising (i) the anti-CTLA-4 antibody fragment disclosed herein and the anti-PD-1 antibody disclosed herein, and a pharmaceutically acceptable carrier, or (ii) the anti-CTLA-4 antibody fragment disclosed herein and the anti-PD-L1 antibody disclosed herein, and a pharmaceutically acceptable carrier.

[0092] The present invention further provides compositions comprising (i) the anti-CTLA-4 antibody fragment and the anti-PD-1 antibody fragment disclosed herein, and a pharmaceutically acceptable vector, or (ii) the anti-CTLA-4 antibody fragment and the anti-PD-L1 antibody fragment disclosed herein, and a pharmaceutically acceptable vector.

[0093] The present invention further provides compositions comprising (i) the anti-CTLA-4 antibody and the anti-PD-1 antibody fragment disclosed herein, and a pharmaceutically acceptable carrier, or (ii) the anti-CTLA-4 antibody and the anti-PD-L1 antibody fragment disclosed herein, and a pharmaceutically acceptable carrier.

[0094] The present invention further provides any one of the anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies or compositions disclosed herein for treating cancer in an individual or for preparing a medicament for treating cancer in an individual.

[0095] The present invention further provides any one of the anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibody fragments or compositions disclosed herein for treating cancer in an individual or for preparing a medicament for treating cancer in an individual.

[0096] In a specific implementation plan, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, kidney cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0097] In a specific implementation plan, the cancer is pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer. Brief description of the attached diagram

[0099] Figure 1A-1E : CTLA-4 blockade-mediated colitis is Fc-dependent. Balb / c mice were treated twice a week with the antibody as shown for 55 days. Figure 1A Profiling analysis of intestinal inflammatory gene expression following administration of Fc-active anti-CTLA-4 antibody (α-CTLA4) or anti-CTLA-4 antibody with D265A substitution effector silencing (α-CTLA4(D265S)). After 7 weeks of treatment twice weekly, proximal small intestine was collected for evaluation of intestinal inflammatory markers by reverse transcription quantitative polymerase chain reaction (PCR). Heat maps showing the fold change in intestinal inflammatory gene expression for both antibodies compared to isotype control treatment are presented. Expression was analyzed across multiple experimental groups, and circulating threshold data were normalized relative to ubiquitin within each experimental group. Normalized data from genes analyzed as part of multiple experimental groups were averaged before determining the fold change compared to isotype control. Figure 1B Weight loss during the experimental period. Figure 1C Intestinal permeability was assessed by measuring FITC-glucan fluorescence in serum on days 49 and 50. Figure 1D The histological findings of the severity of the associated intestinal inflammation and colitis were examined and scored by a qualified pathologist. Figure 1E Representative micrographs of colon histological sections stained with hematoxylin and eosin (H & E) from day 55 onwards.

[0100] Figures 2A-2E : Characterization of CTLA-4 ISVD (nAb) . Figure 2AComparison of effector-silencing CTLA4 ISVD (CTLA4 nAb) and effector-active α-CTLA4; Figure 2B As shown, spleen-activated T cells were cultured for three days in the presence of CLTA4 nAb or α-CTLA4. Proliferation was measured. Figure 2C ), IFNγ ( Figure 2D The production and plotting of IL-2 (Figure 22E) compared to the isotype control (mouse IgG) were compared. 2a The data represents multiples of change from 2 to 3 independent experiments.

[0101] Figures 3A-3D : The combination of CTLA4 nAb and anti-PD-1 antibody (α-PD1) has effective anti-tumor efficacy. . Figure 3A When the tumor reaches 100 mm 3 (range 78-125 mm) 3 When the average tumor size was measured, CT26 tumor-bearing mice received five doses of indicated antibodies (α-CTLA4, α-PD1, α-CTLA4 (D265A)) at 20 mpk every four days and / or CTLA4 nAb at 30 mpk. Data showed the average tumor volume over a 32-day time period. Results represent two independent experiments (n = 10 mice per group). Figure 3B As shown in the figure, the ratio of CD8 T cells / Foxp3+ T cells in the tumor on the first day after treatment. reg Ratio. Results represent two independent experiments (n = 7 mice per group); Figure 3C and Figure 3D Gene expression profile from the entire tumor on day 8 post-treatment. Results represent one or two independent experiments (n = 5 mice per group). *p<0.05, **p<0.01, ***p<0.001 (unpaired t-test). Error bars ± SEM. Figure 3E The figures show the tumor volume of individual animals in each treatment group compared to the isotype control. Complete response (CR) is shown up to day 39 for the reactive treatment group. Data represent two independent experiments in n=10 mice per group.

[0102] Figures 4A-4D : Anti-CTLA-4 antibody-mediated colitis is Fc-dependent. Balb / c mice were treated twice a week for up to 55 days with antibodies (α-CTLA4, α-PD1, α-CTLA4 (D265A)) alone or in combination with CTLA4 nAb, as shown. Figure 4A Weight loss during the experimental period; Figure 4B Histological evaluation of proximal jejunal enteritis at day 55; Figure 4C Photomicrographs of H&E-stained colon histological sections; Figure 4DThe image shows a heatmap illustrating the fold change in intestinal inflammation gene expression in the samples compared to the isotype control treatment. Expression was analyzed across multiple experimental groups, and the circulation threshold data were normalized relative to ubiquitin within each group. Normalized data from genes analyzed as part of multiple experimental groups were averaged before determining the fold change compared to the isotype control.

[0103] Figures 5A-5C : Fc effector function anti-CTLA-4 drives skin inflammation rather than systemic inflammation. Balb / c mice were treated twice a week for 55 days with either α-CTLA4 or α-CTLA4 (D265A) as shown. Figure 5A Photomicrograph of a histological section of ear skin stained with H&E. Figure 5B : Flow cytometry was used to measure the number of IL-17-producing T cells and Foxp3+ T cells in the ear skin. reg The absolute number of cells and neutrophils. Figure 5C Photomicrographs of H&E-stained histological sections of the kidney (top), liver (middle), and lung (bottom). Results represent one of two independent experiments (n = 4–8 mice per group). Scale bar represents 100 µm. Error bars ± SEM.

[0104] Figures 6A-6D : Sufficient Fc-anti-CTLA-4 antibodies do not rule out colonic Foxp3+ T cells. regs . Figure 6A Intracellular CTLA-4 staining in CT26-tumor-bearing mice in the indicated organs. Figure 6B Mean fluorescence intensity (MFI) of CTLA-4 on Foxp3+ Treg cells. **p<0.01, ***p<0.001 (paired t test). Figure 6C and Figure 6D Representative dot plots and statistics of lamina propria of the colon and CT26 tumor infiltration of Foxp3+ Tregs at 24 hours post-treatment are shown. Data represent two to four independent experiments (n=4–12 mice per group). **p<0.01, ***p<0.001 (unpaired t-test). Error bars ± SEM.

[0105] Figures 7A-7D : Fc-function-mediated intestinal damage T in anti-CTLA-4 reg -Mediated colitis inhibition Splenic CD45Rbhigh naive T cells were transferred into CB17-SCID recipient mice and treated with α-CTLA4 or CTLA4 nAb as shown. Figure 7A Weight loss during the experimental period. Figure 7B Photomicrographs of H&E-stained colon histological sections and Figure 7CPathological score on day 47 (n=14-18 mice per group). Figure 7D Gene expression profile of the entire colon on day 47 after naive T cell transfer (n=6 mice per group). Data represent one of two independent experiments. Ns=not significant ****p<0.0001 (unpaired t-test). Error bars ± SEM.

[0106] Figures 8A-8E : FcγR engagement and CTLA-4 blockade activate colon macrophages. Figure 8A and Figure 8B CD16 / CD32 surface expression on macrophages isolated from the spleen, lamina propria of colon, and tumors of mice carrying CT26 was evaluated by flow cytometry. Figure 8C The proportion of macrophages (CD45+CD11b+F4 / 80+) in the spleen, lamina propria of colon, and tumors from mice carrying CT26 was assessed by flow cytometry. Figure 8D : On days 0, 10, and 18 post-treatment, the expression of Il1b, Tnfα, Ifnγ, and Stat1 mRNA was assessed in the colon of mice treated with α-CTLA4, α-CTLA4 (D265A), or CTLA4 nAb. Data represent two independent experiments (n = 8–10 mice per group). ns = not significant, **p < 0.01, ***p < 0.001 (paired t-test). Error bars ± SEM. Figure 8E The proportion of IL-17-producing CD4+ T cells (CD45+TCRb+CD4+CD8a-IL-17A+) in the lamina propria of the colon, the absolute number of IFNγ-producing CD8a+ T cells (CD45+TCRb+CD4-CD8a+IFNγ+), and the number of neutrophils (CD45+CD11b+Ly6G) 高 Measurements were performed by flow cytometry. Results represent one of two independent experiments (n = 4–8 mice per group). **p < 0.01 (unpaired t-test). Scale bar represents 100 µm error bar ± SEM.

[0107] Figures 9A-9B : Antitumor efficacy in mouse syngeneic MB49 bladder tumor model study . Figure 9A When the tumor reaches 102 mm 3 (range 87-117 mm) 3 When the mean tumor size was measured, MB49 tumor-bearing mice received up to four doses of the indicated antibody (30 mg / kg CTLA-4 nAb, 10 mg / kg α-CTLA4, 5 mg / kg α-PD1, or a combination of CTLA4 nAb and α-PD-1) every four days. Data showed the mean tumor volume over a 21-day time period. Results represent two independent experiments (n = 10 mice per group). Figure 9B The figures show the tumor volume of individual animals in each treatment group. Complete response (CR) is shown up to day 21 for the reactive treatment groups. Data are from experiments with n=10 mice per group.

[0108] Figures 10A-10B : Antitumor efficacy in mouse MC38 colon tumor model . Figure 10A When the tumor reaches 220 mm 3 (range 179-261 mm) 3 When the mean tumor size was measured, MC38 tumor-bearing mice were given four doses of the indicated antibody (30 mg / kg CTLA-4 nAb, 10 mg / kg α-CTLA4, 5 mg / kg α-PD1, or a combination of CTLA4 nAb and α-PD-1) every four days. Data showed the mean tumor volume over a 23-day time period. Results represent two independent experiments (n = 10 mice per group). Figure 10B The figures show the tumor volume of individual animals in each treatment group. Complete response (CR) was shown up to day 23 for the responsive treatment group. Data are from experiments with n=10 mice per group.

[0109] Figure 11 : Effector cells induce intestinal inflammation For example, Fc-mediated induction of intestinal inflammation may not depend on T. reg Exclude sketches induced by effector T cells. Invention Details

[0111] definition

[0112] As used herein, “adverse event” or “AE” refers to any adverse and unintentional symptom (including abnormal laboratory findings), condition, or illness temporarily associated with the use of a medical treatment or procedure in an individual, whether or not it is not considered to be related to said medical treatment or procedure, as announced by the U.S. Department of Health and Human Services on November 27, 2017. General adverse events Terminology Standard (CTCAE) Version 5.0The term AE is a unique representation of a specific event used in medical documentation and scientific analysis. Medical treatment may involve one or more AEs, and each AE may have the same or different levels of severity. The severity of AEs is assigned a grade. The CTCAEs are presented in grades 1 to 5, with a unique clinical description of the severity of each AE based on the general guidelines stated above: Grade 1, mild, or asymptomatic or mildly symptomatic, observed only clinically or diagnostically, or not showing a need for intervention; Grade 2, moderate, or minimal, showing a need for local or non-invasive intervention, or limiting age-appropriate instrumental activities of daily living (ADL); Grade 3, severe or medically significant but not immediately life-threatening, or showing a need for hospitalization or prolonged hospitalization, or disability, or limiting ADL; Grade 4, life-threatening consequences or showing a need for urgent intervention; and Grade 5, death associated with the AE.

[0113] As used herein, “antibody” refers to a glycoprotein comprising (a) at least two heavy chains (HCs) and two light chains (LCs) linked by disulfide bonds, or (b) in the case of a camelid antibody, at least two heavy chains (HCs) linked by disulfide bonds. Each HC consists of a heavy chain variable region or domain (V... H It consists of the heavy chain constant region or structural domain. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region contains three structural domains, C... H 1. C H 2 and C H 3. Typically, except for camel antibody species containing only two HCs, the structural unit in the aforementioned cases is a homodimer, and the basic antibody structural unit is a tetramer containing two HC / LC pairs. Each tetramer comprises two identical polypeptide chain pairs, each pair having one LC (approximately 25 kDa) and one HC chain (approximately 50-70 kDa).

[0114] In some naturally occurring antibodies, each light chain contains an LC variable region or domain (V). L ) and LC constant structure domains. An LC constant structure domain contains a structure domain C L Person V H Includes six family members: V H 1. V H 2. V H 3. V H 4. V H 5 and V H 6; and people V L Includes 16 family members: V κ 1. V κ 2. V κ3. V κ 4. V κ 5. V κ 6. V λ 1. V λ 2. V λ 3. V λ 4. V λ 5. V λ 6. V λ 7. V λ 8. V λ 9 and V λ 10. Each of these family members can be further divided into specific subtypes. V H and V L The structural domain can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), dotted with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDR regions and four FR regions arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0115] The variable regions of the heavy and light chains contain binding domains, which enclose CDRs that interact with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The allocation of amino acids to each domain is generally according to… Sequences of Proteins of Immunological Interest , Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv.Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883.

[0116] Generally, amino acid numbering in the heavy chain constant domain begins at position 118, consistent with the Eu numbering scheme. The Eu numbering scheme is based on the amino acid sequence of human IgG1 (Eu), which has constant domains that begin at amino acid position 118 of the IgG1 amino acid sequence described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and for the IgG1, IgG2, IgG3, and IgG4 constant domains, Béranger et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG Created on: 17 / 05 / 2001, Version: 08 / 06 / 2016, it can be viewed at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#r.

[0117] Typically, although the antibody's V H / V L For a system containing six CDRs, V H The three CDRs and V L While there are three CDRs on the heavy chain, the prior art recognizes that, in most cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and instances of generating specific antibodies solely based on the CDR3 of the heavy chain are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000). See also Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (Defining the CDR region of an antibody by sequence); see also Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987) (Defining the CDR region of the antibody by structure).

[0118] exist www.bioinf.org.uk : Prof. Andrew CR Martin's GroupThe following general rules, disclosed in [the literature] and reproduced in the table below, can be used to identify CDRs in antibody sequences that contain amino acids that specifically interact with the epitopes in the antigen to which the antibody binds. Rare examples exist where these generally unchanged features do not appear; however, Cys residues are the most conserved feature.

[0119]

[0120] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two identical polypeptide chain pairs, each pair having an LC (approximately 25 kDa) and an HC (approximately 50-70 kDa) chain. The N-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The C-terminal portion of the HC defines a constant region primarily responsible for the antibody's effector function. Generally, human LCs are classified as κ and λ LCs. Furthermore, human HCs are generally classified as µ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In both LCs and HCs, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, while HCs also include a "D" region of approximately 10 or more amino acids. See also... Fundamental Immunology Chapter 7 (edited by Paul, W., 2nd edition. Raven Press, NY (1989)).

[0121] The heavy chain of an antibody may or may not have a terminal lysine (K) residue, or a terminal glycine and lysine (GK) residue. Therefore, in certain embodiments of the antibody described herein, there are embodiments comprising the heavy chain constant region amino acid sequence shown herein, which further lacks a terminal lysine and terminates with a glycine residue, or in which a terminal glycine residue is also lacking. This is because the terminal lysine, and sometimes glycine and lysine together, may be cleaved during antibody expression or upon introduction into the human body without a significant adverse effect on antibody efficacy, stability, or immunogenicity. In some cases, the nucleic acid molecule encoding the heavy chain may intentionally omit the codon encoding the terminal lysine or the codon encoding both terminal lysine and glycine.

[0122] As used herein, “antibody fragment” or “antigen-binding fragment” refers to a fragment of a full-length antibody, that is, an antibody fragment that retains the ability to bind specifically to an antigen that binds to a full-length antibody but is smaller than the full length and lacks the entire Fc domain or the portions of the Fc domain that confer the binding of the antibody to FcγRs. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; scFv molecules; NANOBODIES; and multispecific antibodies formed from antibody fragments.

[0123] As used herein, a “chimeric antibody” is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein (i) the first and second antibodies are from different species (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)) or (ii) the first and second antibodies are from different isotypes, for example, a variable domain from an IgG1 antibody and a constant domain from an IgG4 antibody. In one aspect, the variable domain is derived from a non-human antibody such as a mouse antibody (“parental antibody”), and the constant domain sequence is derived from a human antibody. In a further aspect, the variable domain is a humanized variable domain derived from a mouse antibody and the constant domain is a human antibody.

[0124] As used herein, “combined treatment” refers to treatment of a human or animal individual, including the sequential or simultaneous administration of a first therapeutic agent and a second therapeutic agent to said individual. Typically, the first and second therapeutic agents are administered separately and not as a mixture; however, there may be embodiments in which the first and second therapeutic agents are mixed prior to administration.

[0125] As used herein, "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), thereby allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al.). Molecular Biology of the Gene (See The Benjamin / Cummings Pub. Co., p. 224 (4th edition) (1987)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conserved substitutions are stated in Table 2.

[0126]

[0127] “Cytotoxic T-lymphocyte-associated antigen-4,” “CTLA-4,” “CTLA4,” “CTLA-4 antigen,” and “CD152” (see, for example, Murata, Am. J. Pathol. 155:453-460 (1999)) are used interchangeably and include variants, isotypes, species homologs of human CTLA-4, and analogs that share at least one epitope with CTLA-4 (see, for example, Balzano, Int. J. Cancer Suppl. 7:28-32 (1992)). The complete CTLA-4 nucleic acid sequence can be found in GenBank under accession number L15006.

[0128] As used herein, “effective function” refers to those biological activities attributable to the antibody’s Fc region and varying with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Antibodies function through many mechanisms, most of which involve binding to other arms of the immune system. Antibodies can simply block molecular interactions, or they can activate the classical complement pathway (known as complement-dependent cytotoxicity or CDC) through the interaction of C1q on the C1 complex with clusters of antibodies. Decisively, antibodies also act as a linker between antibody-mediated and cell-mediated immune responses via Fc receptor binding.

[0129] As used herein, “effecton silencing” refers to an antibody or antibody fragment that exhibits (i) no measurable binding to one or more Fc receptors (FcRs), as measurable in a Biacore assay, wherein the association constant in the micromolar range indicates no measurable binding, or (ii) measurable binding to one or more FcRs, as measurable in a Biacore assay, which is reduced compared to typical binding of the same isotype antibody. In a particular embodiment, the antibody may contain one or more mutations in the HC constant domain and, particularly, the Fc domain, such that the mutated antibody has reduced or no measurable binding to FcγRIIIa, FcγRIIa, and FcγRI compared to a wild-type antibody of the same isotype as said mutated antibody. In certain embodiments, the affinity or association constant of the effector silencing antibody with one or more of FcγRIIIa, FcγRIIa, and FcγRI is reduced to at most 1 / 1000 compared to the affinity with the wild-type isotype; reduced to at most 1 / 1000 to 1 / 100 compared to the affinity with the wild-type isotype; reduced to at most 1 / 100 to 1 / 50 compared to the affinity with the wild-type isotype; or reduced to at most 1 / 50 to 1 / 10 compared to the affinity with the wild-type isotype. In certain embodiments, the effector silencing antibody does not exhibit detectable or measurable binding with one or more of FcγRIIIa, FcγRIIa, and FcγRI compared to binding with the wild-type isotype. Typically, the effector silencing antibody will lack measurable antibody-dependent cytotoxic (ADCC) activity. Effector silencing antibody fragments lack the Fc domain or those portions of the Fc domain that confer binding to FcRs, and therefore will not exhibit detectable or measurable binding to one or more of FcγRIIIa, FcγRIIa, or FcγRI. For effector silencing antibodies or antibody fragments, binding is measured against human FcRs.

[0130] As used in this article, the “Fab fragment” contains a V of LC and an HC. H and C H 1. And excluding the remaining portion of the HC constant structural domain. The C of the Fab molecule. H 1. It cannot form a disulfide bond with another Fab fragment or an HC-containing molecule. The “Fab fragment” can be a papain cleavage product of the antibody.

[0131] As used herein, the “Fab” fragment comprises an LC and an HC fragment, wherein the HC fragment contains V H domain and all the way to C H 1 and C HThe HC constant domain between the two domains and the remaining part of the HC constant domain are excluded, so that interchain disulfide bonds can be formed between the two HCs of the two Fab' fragments to form the F(ab')2 molecule.

[0132] The “F(ab')2 fragment” used in this article contains two LCs and two HC fragments, each HC fragment containing V H domain and all the way to C H 1 and C H The HC constant domain between the two HCs is excluded, thus allowing an interchain disulfide bond to form between the two HCs. The F(ab')2 fragment therefore consists of two Fab' fragments linked by a disulfide bond between the two heavy chains. The F(ab')2 fragment can be obtained by digesting an antibody with pepsin at C60°C. H 1 and C H 2. Site-cutting antibodies between structural domains.

[0133] As used in this article, the "Fc domain" or "Fc" refers to the domain containing the antibody. H 2 and C H 3. Antibodies with three Fc domains can acquire crystallizable segmental domains or regions. In antibodies, the two Fc domains are linked by two or more disulfide bonds and by C... H The Fc domain binds through hydrophobic interactions. The Fc domain can be obtained by digesting the antibody with the protease papain.

[0134] As used in this article, "Fc receptors" or "FcRs" are key immunomodulatory receptors that link antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcµR (IgM), and FcδR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (nanomolar range KD), while FcγRII and FcγRIII are low to intermediate affinity receptors (micromolar range KD). In antibody-dependent cytotoxicity (ADCC), FcRs on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) bind to the Fc region of IgG, which itself binds to the target cell. Binding triggers a signaling pathway that leads to the secretion of various substances, such as hydrolases, perforin, granzymes, and tumor necrosis factor, which mediate the destruction of the target cell. The ADCC effector function levels of human IgG subtypes vary widely. While this is simply dependent on allotype and specific FcR, ADCC effector function is high for human IgG1 and IgG3, but low for IgG2 and IgG4.

[0135] As used in this article, the "Fv region" contains a single V. H and V L Yes, where V H polypeptides and V L Peptides are bound together by disulfide bonds.

[0136] As used in this paper, “humanization” (also known as remodeling or CDR-grafting) is an established technique used to reduce the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (typically rodents) and to improve effector functions (ADCC, complement activation, C1q binding). Artificially engineered mAbs are created using molecular biology techniques; however, simply grafting rodent complementarity-determining regions (CDRs) into human scaffolds often results in a loss of binding affinity and / or specificity of the original mAb. To humanize antibodies, the design of humanized antibodies involves variations in CDR residues, such as conserved amino acid substitutions, and reversion substitutions (reversion mutations) of residues from rodent mAbs into human scaffold regions. Locations can be identified or determined by sequence comparison for structural analysis or by analysis of homology models of the three-dimensional structure of the variable regions. The process of affinity maturation has recently utilized phage libraries to alter the amino acids at selected locations. Similarly, numerous methods have been used to select the most suitable human scaffold in which rodent CDRs are grafted. As datasets of known parameters of antibody structures increase, so do the sophistication and refinement of these techniques. Fragments of the framework sequence from the common or germline sequences of a single antibody, or from the variable regions of each light or heavy chain of several different human mAbs, can be used. Another humanization approach involves modifying the surface residues of purely rodent sequences with the most common residues in human mAbs, a process known as “resurfacing” or “veneering.” Often, human or humanized antibodies are essentially non-immunogenic in humans.

[0137] As used herein, “humanized antibody” refers to an antibody or antibody fragment containing sequences from both human and non-human (e.g., mouse, rat) antibodies. Typically, a humanized antibody will contain at least one, and generally two, variable domains, where hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are those of human immunoglobulin sequences. A humanized antibody may optionally contain at least a portion of the constant regions of human immunoglobulins (e.g., Fc domains).

[0138] As used in this article, "hyperproliferative disease" refers to a condition in which cell growth is increased compared to normal levels. For example, hyperproliferative diseases or conditions include malignant diseases (e.g., esophageal cancer, colon cancer, biliary cancer) and non-malignant diseases (e.g., atherosclerosis, benign prostatic hyperplasia, benign prostatic hyperplasia).

[0139] As used in this article, “immune-related adverse events” or “irAEs” refer to AEs, which are autoimmune manifestations that cause an imbalance in the immune system, such as those that may be attributed to the use of one or more immune restriction point inhibitors, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies.

[0140] As used in this article, “immune response” refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which leads to selective damage, destruction, or elimination from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.

[0141] As used in this article, "immunoglobulin single variable domain" (also known as "ISV" or "ISVD") generally refers to the variable domain of immunoglobulins (which can be a heavy chain or light chain domain, including V...). H V HH or V L (a domain) that can exist without interacting with another variable domain (e.g., without the V domain of a conventional 4-chain monoclonal antibody). H and V L V required between structural domains H / V L Functional antigen-binding sites are formed under conditions of interaction. Examples of ISVDs include NANOBODIES (including V... HH Humanized V HH and / or camel-derived V H For example, camel-derived human V H s), IgNAR, domain, (single domain) antibody (e.g., dAbs™), which is V H The structural domain or derived from V H Domains, and (single-domain) antibodies (e.g., dAbs™), which are V L The structural domain or derived from V L Domains. Based on and / or derived from self-weighted variable domains (e.g., V...). H or V HH ISVDs (with structural domains) are generally preferred.

[0142] As used herein, “monoclonal antibody” refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules constituting the group are identical in amino acid sequence except for the possibility of naturally occurring mutations present in small amounts. In contrast, conventional (polyclonal) antibody formulations generally comprise many different antibodies with different amino acid sequences in their variable domains, often specific to different epitopes. The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by the hybridoma method first described in Kohler et al., Nature 256: 495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). “Monoclonal antibody” can also be isolated from a phage antibody library, for example, using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). See also Presta J. AllergyClin. Immunol. 116:731 (2005).

[0143] As used in this article, “NANOBODY” and “NANOBODIES” are registered trademarks of Ablynx NV.

[0144] As used herein with respect to antibodies or immunoglobulins, “non-human amino acid sequence” refers to an amino acid sequence specific to non-human mammalian amino acid sequences. This term does not include the amino acid sequences of antibodies or immunoglobulins obtained from fully human antibody libraries, where the diversity in the library is generated on a computer chip (see, for example, U.S. Patent Nos. 8,877,688 or 8,691,730).

[0145] "PD-1" refers to programmed death 1 (PD-1). PD-1 CTLA-4 is a repressive member of the extended CD28 / CTLA-4 family of T-cell regulators (Okazaki et al., Curr. Opin. Immunol. 14: 391779-82 (2002); Bennett et al., J. Immunol. 170:711-8 (2003)). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. PD-1The gene encodes a 55 kDa type I transmembrane protein (Agata et al., Intl. Immunol. 8:765-72(1996)). Two ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified and have shown to interact with... PD-1 This combination downregulates T cell activation (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 Immunosuppressive proteins known as negative regulators of TCR signaling (Ishida, Y. et al., EMBO J. 11:3887-3895 (1992); Blank, C. et al., Immunol. Immunother. 56(5):739-745 (Epub 29 Dec 2006)). The interaction between PD-1 and PD-L1 can act as an immune limiting point, which can lead to, for example, a reduction in tumor-infiltrating lymphocytes, a reduction in T-cell receptor-mediated proliferation, and / or immune evasion by cancer cells (Dong et al., J. Mol. Med. 81:281-7 (2003); Blank et al., Cancer Immunol. Immunother. 54:307-314 (2005); Konishi et al., Clin. Cancer Res. 10:5094-100 (2004)). This can be mitigated by inhibiting... PD-1 Local interaction with PD-L1 or PD-L2 to reverse immunosuppression; when PD-1 When the interaction with PD-L2 is also blocked, the effects are additive (Iwai et al., Proc. Nat'l. Acad. Sci. USA99:12293-12297 (2002); Brown et al., J. Immunol. 170:1257-66 (2003)).

[0146] “Programmed cell death 1”, “Programmed cell death 1”, “Protein PD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1”, and “hPD-1” are used interchangeably and include variants, isotypes, species homologs of human PD-1, and analogs that share at least one epitope with PD-1. The complete PD-1 sequence can be found in GenBank under accession number U64863.

[0147] As used herein, “ScFv” or “single-chain variable region fragment” refers to a V-shaped structure containing short linker peptides fused or linked together by 10 to approximately 25 amino acids.H and V L The fusion protein. The linker is typically rich in glycine for flexibility, and serine or threonine for solubility, and can transfer V... H of N -Terminal and V L of C - Terminal connections, or vice versa. Despite the removal of the constant region and the introduction of adapters, the protein retains the specificity of the original immunoglobulin.

[0148] As used herein, “subtherapeutic dose” means a dose of a therapeutic compound (e.g., an antibody) that is lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disease (e.g., cancer). The dose of a therapeutic compound may vary depending on the disease being targeted. For example, a subtherapeutic dose of a CTLA-4 antibody is a single dose of less than about 3 mg / kg of the antibody, i.e., a known monotherapy dose of the anti-CTLA-4 antibody YERVOY for the treatment of unresectable or metastatic melanoma, or a single dose of less than about 10 mg / kg of YERVOY, a known monotherapy dose for adjuvant melanoma.

[0149] As used herein, “treat” or “treating” refers to the internal or external administration of a therapeutic agent, such as a composition containing any antibody or antigen-binding fragment of the present invention, to a subject or patient having one or more symptoms of a disease or suspected of having a disease, which has therapeutic or preventative activity against the disease. Generally, the agent is administered in an amount that effectively reduces one or more symptoms of a disease in the treated subject or population, whether by inducing the resolution of one or more such symptoms to any clinically measurable extent or by inhibiting the progression of one or more such symptoms to any clinically measurable extent. The amount of a therapeutic agent that effectively reduces any particular disease symptom may vary depending on factors such as the patient’s disease state, age, and weight, and the ability of the drug to elicit the desired response in the subject. Whether the disease symptoms have been reduced can be assessed by any clinical measurement generally used by a physician or other skilled healthcare provider to assess the severity or progression of the disease symptoms. The term further includes delaying the development of symptoms associated with the condition and / or reducing the severity of symptoms of the condition. The term further includes improving existing uncontrolled or unwanted symptoms, preventing additional symptoms, and improving or preventing the underlying cause of such symptoms. Therefore, the term means that a beneficial result has been given to a human or animal subject suffering from a condition, disease, or symptom, or having the potential to develop such a condition, disease, or symptom.

[0150] As used herein, “therapeutic effective amount” refers to the amount of a specific substance sufficient to achieve the desired effect in the treated subject. For example, this might be the amount of CTLA-4 blocker necessary to inhibit CTLA-4 activation and induce an antitumor response, or the amount necessary to enhance anti-PD-1 or PD-L1 responsiveness when co-administered with anti-PD-1 or anti-PD-L1 blockers, respectively.

[0151] The “therapeutic index,” also known as the “therapeutic window,” “safety window,” or “therapeutic ratio,” as used in this article, is a comparison of the amount of therapeutic agent that produces a therapeutic effect to the amount of therapeutic agent that causes toxicity.

[0152] As used herein, “treatment” when applied to human or veterinary individuals means therapeutic treatment, which includes contact between an antibody or antigen-binding fragment and a human or animal individual requiring treatment with said antibody or antibody fragment.

[0153] As used in this article, “V” HH " indicates V H The domains are obtained from, originate from, or are derived from HC antibodies. Heavy chain antibodies are functional antibodies with two HCs and no LCs. Heavy chain antibodies are found in and are available from camel family animals (Camelidae). Camelidae (members of)

[0154] Introduction

[0155] PD-1 antagonists, such as the commercially available anti-PD-1 antibodies Keytruda and Opdivo, contain a human IgG4 backbone with reduced FcγR function. This is because preclinical studies using anti-PD-1 antibodies with FcγR binding function have shown weak antitumor efficacy attributable to the exclusion of CD8+ cytotoxic T cells (CTLs), which are essential for tumor immunotherapy (see, for example, International Patent Application WO2014 / 089113). In contrast, preclinical studies have shown that monotherapy with anti-CTLA-4 antibodies requires FcγR function to achieve a strong antitumor response, and these preclinical studies used mouse IgG4 antibodies with high FcγR-binding affinity. 2aAnti-CTLA-4 antibodies were compared with mutant mouse IgG1-anti-CTLA-4 antibodies lacking measurable FcγR-binding affinity (see, for example, Selby et al., Cancer Immunol Res. 1:32-42 (2013). The need for FcγR function in anti-CTLA-4 antagonist monotherapy is similar to that in mouse tumor models attributed to T cells in the spleen or lymph nodes. reg T regulatory cells (T cells) with higher CTLA-4 expression on TILs compared to TILs reg The exclusion of related factors (Simpson et al., J. Exp. Med. 210:1695-710 (2013)).

[0156] The inventors of this invention hypothesize that the need for FcγR function to be effective against anti-CTLA-4 antibodies can be circumvented by combining anti-CTLA-4 antibodies with anti-PD-1 antibodies. This hypothesis is supported by emerging data illustrating CD28-mediated co-stimulation in anti-PD-1-mediated CD8 depletion. + Key roles in the activation of cytotoxic T cells. Anti-CTLA-4 and anti-PD-1 antibodies exert their antitumor activity through different mechanisms. Importantly, the combined effect of anti-CTLA-4 and anti-PD-1 antibodies is not merely additive, because the combined blocking effect of these two antibodies leads to the activation of a large number of genes, including proliferation-related and chemokine genes, which are not activated by either antibody alone (see, for example...). Figure 3C and 4D These data suggest that the mechanism of action of CTLA-4 blockade in monotherapy differs from its mechanism of action when combined with PD-1 blockade.

[0157] Emerging data indicate that CD28-mediated co-stimulation activates the T(T) effect after PD-1 blockade. eff The importance of PD-1 signaling in cells. PD-1 signaling dephosphorylates CD28, rather than TCR as previously assumed, and the enhanced antitumor response observed after PD-1 blockade requires CD28 signaling. Therefore, while monotherapy CTLA-4 blockade may primarily target T cell sensitization events, combining CTLA-4 blockade with PD-1 blockade to promote the activation of exhausted T cells is expected to exceed what PD-1 blockade alone would anticipate. The inventors hypothesize that the mechanism is enhanced by CTLA-4 antagonists, resulting in increased CD28-mediated activation independent of Fc receptor function and T cells that may play an important role in irAE-mediated toxicity. reg The exclusion of cells becomes possible.

[0158] Therefore, a potential warning for anti-CTLA-4 antibodies (Fc-functional antibodies) that bind to FcRs is T. reg Exclusion or cellular bridging of myeloid cells with T cells may induce undesirable immune-related inflammation. The inventors hypothesize that Fc function may be one of the reasons for the observed irAEs associated with CTLA-4-blocking cancer immunotherapy. One criticism that has been used to oppose the potential role of Fc function in inducing irAEs is that treatment with ipilimumab (on the human IgG1 backbone) and trimemumab (on the human IgG2 backbone) is associated with intestinal inflammation. Although the human IgG2 Fc domain has a significantly lower affinity for human FcγRs compared to human IgG1, direct comparisons of antibodies with human IgG2 and IgG1 backbones have shown that both evoke similar levels of Fc function using in vitro ADCC and ADCP bioassays (e.g., Vargas et al., Cancer Cell. 33:649-663 (2018)). Furthermore, in vivo T cells with chimeric anti-mouse CTLA-4 antibodies with either a human IgG1 or human IgG2 isotype backbone have shown similar levels of Fc function. reg The exclusion and antitumor activities were the same in human FcγR knock-in mice (Vargas et al., ibid.).

[0159] A key obstacle to assessing the potential role of Fc function in inducing intestinal inflammation in syngeneic tumor models is the lack of measurable inflammation and colitis when using mouse anti-CTLA-4 alternative antibodies. To circumvent this obstacle, the inventors have employed a PCR-based experimental subject group previously developed by Cayate et al., Clin. Transl. Gastroenterol. 3: e10 (2012) to measure the upregulation of intestinal inflammatory genes associated with inflammatory bowel disease (IBD) in a mouse IBD model. As illustrated in the examples herein, this PCR-based experimental subject group enabled the inventors to detect increased expression of biomarker genes indicative of intestinal inflammation in mice treated with an Fc-functional anti-mouse CTLA-4 antibody (α-CTLA4), even in the absence of obvious histological evidence of colitis or tissue damage (see [link to documentation]). Figure 1A , 3C The observations of subclinical stimulation of intestinal inflammatory gene expression pathways, along with 4D, inspired the inventors to extend the treatment course to determine whether underlying inflammation would progress to the development of clinical colitis. The irAEs colitis mouse model enabled the inventors to run empirical experiments to assess the need for Fc function in inducing intestinal inflammation in syngeneic tumor models used in preclinical immuno-oncology development, both in the presence and absence of an accompanying antitumor response.

[0160] The results described in the examples clearly demonstrate that, in the monotherapy setting, neither the effector-silencing anti-CTLA-4 antibody nor the effector-silencing anti-CTLA-4 antibody fragment elicited measurable antitumor activity. However, administration of the effector-silencing antibody or the effector-silencing antibody fragment in combination with the anti-PD-1 antibody resulted in antitumor activity similar to that elicited by the effector-functional anti-CTLA-4 antibody alone or in combination with the anti-PD-1 antibody (see Figure 3A), and there were no intestinal or skin irAEs observed for the effector-functional anti-CTLA-4 antibody alone or in combination with the anti-PD-1 antibody. Figure 4B and 5A ) or weight loss ( Figure 4A Given these results and the potential T cells associated with Fc-mediated antitumor activity and intestinal inflammation, reg The invention, based on the discovery of the non-dependent mechanism, enables CTLA-4 / PD-1 blocking combined with anti-cancer immunotherapy with improved therapeutic index and wider application.

[0161] Combination therapy

[0162] The present invention provides a combination therapy for cancer, comprising administering to an individual in need of cancer therapy (i) a PD-1 blocking agent selected from anti-PD-1 antibody, anti-PD-L1 antibody, effector-silencing anti-PD-1 antibody, effector-silencing anti-PD-L1 antibody, effector-silencing anti-PD-1 antibody fragment, and effector-silencing anti-PD-L1 antibody fragment; and (ii) an effector-silencing CTLA-4 blocking agent selected from effector-silencing anti-CTLA-4 antibody and effector-silencing anti-CTLA-4 antibody fragment.

[0163] The effector silencing CTLA-4 blocker can be administered in combination therapy with a PD-1 blocker at a dose greater than the 1 mg / kg subtherapy dose of ipilimumab / nivolumab in combination therapy for advanced renal cell carcinoma or microsatellite instability-high or mismatch repair deficient metastatic colorectal cancer approved by the US FDA, and has a lower induction rate than observed with ipilimumab / nivolumab combination therapy. General adverse events Terminology Standard (CTCAE) Version 5.0 The risk of grade 1-2 cutaneous or intestinal irAEs as stated in the description is for at least a portion of the duration of combination therapy or the period during which the individual is receiving combination therapy. In a specific embodiment, the dose does not induce grade 1 irAEs for at least a portion of the duration of combination therapy or the period during which the individual is receiving combination therapy.

[0164] Therefore, in certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose greater than 1 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose of at least 3 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose of at least 10 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose of at least 15 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose of at least 20 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a dose from 3 mg / kg to 20 mg / kg. In certain embodiments, the effector-silencing CTLA-4 blocker can be administered to an individual at a fixed dose, independent of individual weight, such as greater than 100 mg.

[0165] In a specific embodiment of combination therapy, the effector-silencing CTLA-4 blocker is either an effector-silencing anti-CTLA-4 antibody or (b) an effector-silencing anti-CTLA-4 antibody fragment. Because effector functional activity of the anti-CTLA-4 antibody is not required, the anti-CTLA-4 antibody either has an HC domain that has been artificially modified to be "effector-silencing," i.e., its Fc domain is modified to have reduced or no measurable FcR binding compared to the Fc domain of a wild-type antibody of the same isotype as the effector-silencing antibody (e.g., the Fc domain of a non-mutated IgG1, IgG2, IgG3, or IgG4 Fc domain), as determined by a Biacore assay. The effector-silencing anti-CTLA-4 antibody fragment either lacks an Fc domain or lacks those regions of the Fc domain that bind one or more FcRs.

[0166] In certain embodiments, the combined treatment of the present invention is administered to an individual before or after surgery to remove a tumor and can be used before, during or after radiotherapy.

[0167] In certain embodiments, the combination therapy of the present invention is administered to an individual who has not previously been treated with a biological therapy or chemotherapy agent, i.e., the individual is untreated. In other embodiments, the combination therapy is administered to an individual who has failed to achieve a sustained response following prior treatment with a biological therapy or chemotherapy agent, i.e., the individual has undergone treatment.

[0168] In certain embodiments, the combined treatment of the present invention is used to treat tumors large enough to be detected by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scans. In some embodiments, the combined treatment of the present invention is used to treat tumors with a diameter of at least about 200 mm. 3 300 mm 3 400 mm 3 500 mm 3 750 mm 3 Or up to 1000 mm 3 Late-stage tumors of that size.

[0169] In certain embodiments, the combination therapy of the present invention is administered to an individual with cancer that is positive for PD-L1 expression. In some embodiments, PD-L1 expression is detected using a diagnostic anti-human PD-L1 antibody or its antigen-binding fragment in a fixed formalin-embedded (FFPE) or frozen tissue section taken from the individual. Before initiating treatment with the combination therapy of the present invention, the individual's physician may schedule diagnostic testing to determine PD-L1 expression in a tumor tissue sample taken from the individual, but it is anticipated that the physician may schedule the first or subsequent diagnostic testing at any time after the start of treatment, such as, for example, after the completion of a treatment cycle.

[0170] Combination therapy may include any of the exemplary effector-silencing anti-CTLA-4 antibodies or effector-silencing anti-CTLA-4 antibody fragments disclosed herein, in combination with any of the exemplary anti-PD-1 antibodies or anti-PD-1 antibody fragments disclosed herein or any of the exemplary anti-PD-L1 antibodies or anti-PD-L1 antibody fragments disclosed herein.

[0171] (a) Effector silencing antibody

[0172] The effector silencing antibody of the present invention comprises an HC constant domain or its Fc domain, which has been modified such that, compared with an unmodified antibody of the same IgG isotype, the antibody does not exhibit measurable binding to one or more FcRs, or exhibits reduced binding to one or more FcRs. In a further embodiment, the effector silencing antibody may not exhibit measurable binding to each of FcγRIIIa, FcγRIIa, and FcγRI, or exhibit reduced binding to each of FcγRIIIa, FcγRIIa, and FcγRI, compared with an unmodified antibody of the same IgG isotype. In a particular embodiment, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

[0173] In a particular embodiment, the effector silencing antibody comprises an asparagine (Asn) residue modified to lack position 297 (Eu numbering system) of the HC constant domain. N - Glycosylated Fc domains of IgG1 or IgG2, IgG3 or IgG4 isoforms. N -The common sequence for glycosylation is Asn-Xaa-Ser / Thr (where Xaa at position 298 is any amino acid except Pro); in all four isotypes N - The glycosylation concordance sequence is Asn-Ser-Thr. This modification can be achieved by replacing the codon encoding Asn at position 297 in a nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, such as Ala, Asp, Gln, Gly, or Glu. For example, N297A, N297Q, N297G, N297E, or N297D. Alternatively, the codon for Ser at position 298 can be replaced with a codon for Pro, or the codon for Thr at position 299 can be replaced with any codon other than the codon for Ser. In further alternatives, the constitutive... N Each amino acid in the glycosylated concordant sequence is replaced by another amino acid. This modified IgG molecule has no measurable effector function. In a particular embodiment, these mutant HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations. In a further embodiment, it is modified to lack the amino acid at position 297. N - This glycosylated IgG may further include one or more additional mutations disclosed herein to eliminate measurable effector functions.

[0174] An exemplary IgG1 HC constant domain mutation at position 297 eliminates the HC constant domain. N - Glycosylation, as shown in SEQ ID NO:44, is an exemplary IgG2 HC constant domain mutated at position 297, which eliminates the HC constant domain. N - Glycosylation, shown in SEQ ID NO:50, and an exemplary IgG4 HC constant domain mutated at position 297, which eliminates the HC constant domain. N - Glycosylation, shown in SEQ ID NO:56. In a particular embodiment, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0175] In a particular embodiment, the Fc domain of the HC constant domain of IgG1, IgG2, IgG3, or IgG4 constituting the effector-silencing antibody is modified with one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the position is identified according to the Eu number), and wherein said HC constant domain is effector-silenced. In a particular embodiment, the modified IgG1 further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0176] In a particular embodiment, the HC constant domain comprises substitutions of L234A, L235A, and D265S (wherein the positions are identified according to Eu designations). In a particular embodiment, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu designations). These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831; U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; and Sondermann et al., Nature 406, 267-273 (July 20, 2000).

[0177] In the specific embodiments described above, the HC constant domain comprises L234A / L235A / D265A; L234A / L235A / P329G; L235E; D265A; D265A / N297G; or V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions, wherein the positions are identified according to the Eu number. In the specific embodiments, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0178] In a particular embodiment, the effector-silencing antibody comprises an IgG1 isotype wherein the Fc domain of the HC constant domain has been modified to be effector-silenced by replacing amino acids at positions 233 to 236 of IgG1 with amino acids from the corresponding human IgG2 HC and amino acids at positions 327, 330, and 331 with amino acids from the corresponding human IgG4 HC, wherein said positions are identified according to Eu numbering (Armour et al., Eur. J. Immunol. 29(8):2613-24 (1999); Shields et al., J. Biol. Chem. 276(9):6591-604 (2001)). In a particular embodiment, the modified IgG1 further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0179] In a particular embodiment, the effector silencing antibody comprises a V protein fused to or linked to the HC constant domain of a hybrid human immunoglobulin. H Structural domain, which is based on N -End to C - The terminal direction includes a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region contains at least a portion of the amino acid sequence of the human IgD hinge region or the human IgG1 hinge region; and the CH2 domain is the human IgG4 CH2 domain, a portion of which is located within its... N -The terminal region is bordered by human IgG2 CH2 or human IgD CH2 domains. N - Replacement of 4-37 amino acid residues in the terminal region. This heterozygous human HC constant domain is disclosed in U.S. Patent No. 7,867,491, the entirety of which is incorporated herein by reference.

[0180] In a particular embodiment, the effector silencing antibody comprises an IgG4 HC constant domain, wherein the serine at position 228 according to the Eu system is replaced by a proline, see, for example, SEQ ID NO:52. This modification prevents the formation of a potential interchain disulfide bond between cysteines at positions Cys226 and Cys229 in the EU system, which could interfere with the formation of a proper intrachain disulfide bond. See Angal et al., Mol. Imunol. 30:105 (1993); see also (Schuurman et al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NOs: 14 and 41). In a further embodiment, in addition to the S228P substitution, the IgG4 constant domain also includes amino acid substitutions of P239G, D265A, or D265A / N297G, wherein the positions are identified according to the Eu number. In the particular embodiment described above, the IgG4 HC constant domain is the human HC constant domain. In a particular embodiment, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved mutations or non-conserved mutations.

[0181] An exemplary IgG1 HC constant domain includes an HC constant domain comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. An exemplary IgG2 HC constant domain has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. An exemplary IgG4 HC constant domain has an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0182] More specific examples of effector silencing antibodies are described below in conjunction with specific exemplary effector silencing anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-1 antibodies.

[0183] (b) Exemplary effector-silencing anti-CTLA-4 antibody

[0184] Exemplary effector-silencing anti-CTLA-4 antibodies and compositions comprising these antibodies that can be used in combination therapies of the present invention include any effector-silencing anti-CTLA-4 antibody that binds to CTLA-4 and inhibits CTLA-4 binding to B7. Specific effector-silencing anti-CTLA-4 antibodies include the following effector-silencing anti-CTLA-4 antibodies and compositions comprising any one of these antibodies and a pharmaceutically acceptable carrier.

[0185] In a particular implementation, the effector-silencing anti-CTLA-4 antibody comprises (i) a V-CDR of ipilimumab comprising three HC-CDRs fused to or linked to an HC constant domain. H The HC constant domain does not exhibit measurable binding to FcγRIIIA, FcγRIIA, and FcγRI, or exhibits reduced binding, as determined by Biacore assays, compared to peptides containing wild-type IgG constant domain regions, and (ii) V of the three LC-CDRs containing ipilimumab fused to or linked to the LC κ or λ constant domain. L The three HC-CDRs contain SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and the three LC-CDRs contain SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0186] In a further embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) a V-CDR of trimexumab containing three HC-CDRs fused to or linked to the HC constant domain. H The HC constant domain does not exhibit measurable binding to FcγRIIIA, FcγRIIA, and FcγRI, or exhibits reduced binding, as determined by Biacore assays, compared to peptides containing wild-type IgG constant domain regions, and (ii) V of the three LC-CDRs containing trimexumab fused to or linked to the LC κ or λ constant domain. L The three HC-CDRs contain SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively, and the three LC-CDRs contain SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively.

[0187] In a further embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) ipilimumab V H and V L Domain, (ii) V of trimelimumab H and V L Structural domain, (iii) V of REGN4659H and V L Structural domain, (iv) V of AGEN1884w H and V L The domain, or (v) the anti-CTLA-4 antibody clone 2C8 disclosed in International Patent Application WO2017194265. H and V L Domain. Ipilimumab V H The domain contains the amino acid sequence shown in SEQ ID NO:7, and V L The domain contains the amino acid sequence shown in SEQ ID NO:8. Trimelimumab V H The domain contains the amino acid sequence shown in SEQ ID NO:15, and V L The domain contains the amino acid sequence shown in SEQ ID NO:16. REGN4659 V H The domain contains the amino acid sequence shown in SEQ ID NO:95, and V L The domain contains the amino acid sequence shown in SEQ ID NO: 96. AGEN1884w V H The domain contains the amino acid sequence shown in SEQ ID NO:97, and V L The domain contains the amino acid sequence shown in SEQ ID NO:98. In a particular embodiment, V H and V L The domain further includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0188] In a further embodiment, the effector-silencing anti-CTLA-4 antibody comprises 8D2 / 8D2(RE) (see U.S. Patent Application Publication No. 20170216433 and International Application WO2018183408), 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15, or 8D2H2L17 V H and V L Structural domain, where V H The domain is fused to or linked to the HC constant domain, which, compared to peptides containing wild-type IgG constant domain regions, does not exhibit measurable binding to FcγRIIIA, FcγRIIA, and FcγRI or exhibits reduced binding, as determined by Biacore assays, and V L The structural domain is fused or connected with the LC κ or λ constant structural domain.

[0189] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody comprises a variant of 8D2 / 8D2(RE), 8D2H1L1, 8D2H2L2, 8D2H2L15, or 8D2H2L17, wherein the V of said variant H The methionine at position 18 in the amino acid sequence is replaced by isoleucine. Therefore, effector-silencing anti-CTLA-4 antibodies can contain the V of 8D2 / 8D2(RE)-variant 1. H and V L V of 8D2H1L1-variant 1 H and V L V of 8D2H2L2- variant 1 H and V L V of 8D2H2L15-variant 1 H and V L Or V of 8D2H2L17-variant 1 H and V L .

[0190] In a further embodiment, the effector-silencing anti-CTLA-4 antibody has (i) a V comprising the amino acid sequence shown in SEQ ID NO:73. H The domain and V containing the amino acid sequence shown in SEQ ID NO:74 L (ii) a VH domain containing the amino acid sequence shown in SEQ ID NO:75 and a V domain containing the amino acid sequence shown in SEQ ID NO:76. L (iii) a domain containing the amino acid sequence shown in SEQ ID NO:77. H The domain and V containing the amino acid sequence shown in SEQ ID NO:78 L (iv) V containing the amino acid sequence shown in SEQ ID NO:79 H The domain and V containing the amino acid sequence shown in SEQ ID NO:80 L Domain; (v) V containing the amino acid sequence shown in SEQ ID NO:81 H The domain and V containing the amino acid sequence shown in SEQ ID NO:82 L Domain; (vi) V containing the amino acid sequence shown in SEQ ID NO:83 H The domain and V containing the amino acid sequence shown in SEQ ID NO:84 L Domain; (vii) V containing the amino acid sequence shown in SEQ ID NO:85 H The domain and V containing the amino acid sequence shown in SEQ ID NO:86L (viii) A domain containing the amino acid sequence shown in SEQ ID NO:87. H The domain and V containing the amino acid sequence shown in SEQ ID NO:88 L Domain; (ix) V containing the amino acid sequence shown in SEQ ID NO:89 H The domain and V containing the amino acid sequence shown in SEQ ID NO:90 L Domain; (x) V containing the amino acid sequence shown in SEQ ID NO:91 H The domain and V containing the amino acid sequence shown in SEQ ID NO:92 L Domain; or (xi) containing the amino acid sequence shown in SEQ ID NO:93. H The domain and V containing the amino acid sequence shown in SEQ ID NO:94 L Domain. In a particular implementation, V H and V L The domain further includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0191] In a further implementation of the effector silencing anti-CTLA-4 antibody, V H The domain is fused to or linked to the IgG4 HC constant domain or to an IgG1, IgG2, or IgG4 HC constant domain that has been modified to include one or more mutations to silence the resulting anti-CTLA4 antibody effector.

[0192] In one embodiment, the effector-silencing anti-CTLA-4 antibody comprises an IgG1 Fc domain having (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylated, or further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions of the Fc domain; (ii) amino acid substitution mutations selected from N297A, L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, N297A / D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A / N297G, or further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions of the Fc domain; Or a mutant Fc domain with 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from L234A / L235A / D265A, L234A / L235A / P329G, L235E, D265A, E233A / L235A, S267E / L328F, S2339D / A330L / I332E, L235G / G236R, D356E / L358M, L234F / L235E / P331S / D365E / L358M and D265A, or further containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0193] In another embodiment, the effector-silencing anti-CTLA-4 antibody comprises an IgG2 Fc domain having (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylated, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further comprising a mutation containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - Glycosylation, and amino acid substitution mutations selected from N297A / D265S, D265A, P329G / D265A / N297G or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0194] In a further embodiment, the effector-silencing anti-CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution, and further comprises (i) starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N - A glycosylated Fc domain, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an Fc domain selected from N267A, P329G, and D265A / N297A, or a Fc domain further comprising a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an Fc domain starting from amino acid position 297. N - Mutations at the glycosylation site Asn-Xaa-Ser / Thr, which eliminate the mutations in the... N - Glycosylation site N- Glycosylation, and amino acid substitution mutations selected from N267A, P329G and D265A / N297A, or further including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions in the Fc domain, wherein the amino acid positions in (i), (ii) and (iii) are identified according to the Eu number.

[0195] Table 4-18 provides specific exemplary anti-CTLA-4 antibodies that can be used in combination with anti-PD-1 or anti-PD-L1 antibodies in a therapeutic context to treat individuals with cancer. The invention also provides antibodies shown in the table other than ipilimumab consisting solely of N297A substitutions, and compositions other than those comprising ipilimumab consisting solely of N297A substitutions, each composition comprising the antibody shown in the table and a pharmaceutically acceptable carrier. All HC amino acid substitution positions in Table 4-18 are according to the Eu numbering scheme.

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] (c) Exemplary effector-silencing anti-CTLA-4 antibody fragment

[0206] Exemplary effector-silencing anti-CTLA-4 antibody fragments and compositions comprising them that can be used in combination therapies of the present invention include any antibody fragment that binds to CTLA-4 and inhibits CTLA-4 binding to B7. Specific examples of such anti-CTLA-4 antibody fragments include the following anti-CTLA-4 antibody fragments and compositions, each composition comprising an effector-silencing anti-CTLA-4 antibody fragment and a pharmaceutically acceptable carrier.

[0207] In a particular implementation, the effector-silencing anti-CTLA-4 antibody fragment is Fv, scFv, F(ab), or F(ab')2, which comprises (i) a V-CDR containing three HC-CDRs of ipilimumab.H (ii) V containing three LC-CDRs of ipilimumab L The three HC-CDRs contain SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the three LC-CDRs contain SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 7 respectively.

[0208] In a specific implementation, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) a V-CDR containing three HC-CDRs of trimexumab. H (ii) V containing three LC-CDRs of trimemumab L The three HC-CDRs contain SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively, and the three LC-CDRs contain SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively.

[0209] In a specific implementation, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) the V of ipilimumab. H and V L Domain, (ii) V of trimelimumab H and V L Structural domain, (iii) V of REGN4659 H and V L Structural domain, (iv) V of AGEN1884w H and V L The domain, or (v) the anti-CTLA-4 antibody clone 2C8 disclosed in International Patent Application WO2017194265. H and V L Domain. Ipilimumab V H The domain contains the amino acid sequence shown in SEQ ID NO:7, and V L The domain contains the amino acid sequence shown in SEQ ID NO:8. Trimelimumab V H The domain contains the amino acid sequence shown in SEQ ID NO:15, and V L The domain contains the amino acid sequence shown in SEQ ID NO:16. REGN4659 V H The domain contains the amino acid sequence shown in SEQ ID NO:95, and V L The domain contains the amino acid sequence shown in SEQ ID NO: 96. AGEN1884w V HThe domain contains the amino acid sequence shown in SEQ ID NO:97, and V L The domain contains the amino acid sequence shown in SEQ ID NO:98.

[0210] In a particular implementation, the effector-silencing anti-CTLA-4 antibody fragment contains the V of ipilimumab. H and V L Trimelimumab V H and V L REGN4659's V H and V L ,AGEN1884w's V H and V L V of 8D2 / 8D2 (RE) H and V L 8D2H1L1 of V H and V L V of 8D2H2L2 H and V L 8D3H3L3 of V H and V L 8D2H2L15 of V H and V L Or V of 8D2H2L17 H and V L .

[0211] In a particular embodiment, the anti-CTLA-4 antibody or anti-CTLA-4 antibody fragment contains the V of 8D2 / 8D2(RE)-variant 1. H and V L V of 8D2H1L1-variant 1 H and V L V of 8D2H2L2- variant 1 H and V L V of 8D2H2L15-variant 1 H and V L Or V of 8D2H2L17-variant 1 H and V L .

[0212] In a particular embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) a V containing the amino acid sequence shown in SEQ ID NO:73. H The domain and V containing the amino acid sequence shown in SEQ ID NO:74 L (ii) a VH domain containing the amino acid sequence shown in SEQ ID NO:75 and a V domain containing the amino acid sequence shown in SEQ ID NO:76. L(iii) a domain containing the amino acid sequence shown in SEQ ID NO:77. H The domain and V containing the amino acid sequence shown in SEQ ID NO:78 L (iv) V containing the amino acid sequence shown in SEQ ID NO:79 H The domain and V containing the amino acid sequence shown in SEQ ID NO:80 L Domain; (v) V containing the amino acid sequence shown in SEQ ID NO:81 H The domain and V containing the amino acid sequence shown in SEQ ID NO:82 L Domain; (vi) V containing the amino acid sequence shown in SEQ ID NO:83 H The domain and V containing the amino acid sequence shown in SEQ ID NO:84 L Domain; (vii) V containing the amino acid sequence shown in SEQ ID NO:85 H The domain and V containing the amino acid sequence shown in SEQ ID NO:86 L (viii) A domain containing the amino acid sequence shown in SEQ ID NO:87. H The domain and V containing the amino acid sequence shown in SEQ ID NO:88 L Domain; (ix) V containing the amino acid sequence shown in SEQ ID NO:89 H The domain and V containing the amino acid sequence shown in SEQ ID NO:90 L Domain; (x) V containing the amino acid sequence shown in SEQ ID NO:91 H The domain and V containing the amino acid sequence shown in SEQ ID NO:92 L Domain; or (xi) containing the amino acid sequence shown in SEQ ID NO:93. H The domain and V containing the amino acid sequence shown in SEQ ID NO:94 L Structural domain.

[0213] In a particular implementation, the effector-silencing anti-CTLA-4 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD containing a variable domain (V) of camel-only heavy chain antibodies. HHHowever, the ISVDs described herein must not contain the following ISVDs as disclosed in International Patent Applications WO2008071447, WO2017087587 and WO2017087588: CDR1 containing the amino acid sequence FYGMG (SEQ ID NO: 69), CDR2 containing the amino acid sequence DIRTSAGRTTYADSVKG (SEQ ID NO: 70), and CDR3 containing the amino acid EMSGISGWDY (SEQ ID NO: 71) or EPSGISGWDY (SEQ ID NO: 72), and ISVD variants containing one, two or three mutations in CDR3 as disclosed in WO2008071447. ISVDs containing the CDRs are not excluded from the conditions only in embodiments where one or more ISVDs are fused to or linked with an effector silencing antibody constant domain or Fc domain, such as any effector silencing antibody constant or Fc domain disclosed herein.

[0214] (d) Exemplary anti-PD-1 antibody

[0215] Exemplary anti-PD-1 antibodies that can be used in combination therapies of the present invention include any antibody that binds to PD-1 and inhibits PD-1 binding to PD-L1. In a further embodiment, the exemplary anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and cemiplimab-rwlc. Exemplary antibodies include the following anti-PD-1 antibodies and compositions comprising an anti-PD1 antibody and a pharmaceutically acceptable salt.

[0216] Pembrolizumab, also known as Keytruda, lambolizumab, MK-3475, or SCH-900475, is a humanized anti-PD-1 antibody disclosed in U.S. Patent Nos. 8,354,509 and WO2009 / 114335 and in, for example, Hamid et al., New England J. Med. 369 (2): 134-144 (2013). The heavy and light chains of pembrolizumab are shown by the amino acid sequences illustrated in SEQ ID NOs: 27 and 28, respectively.

[0217] Nivolumab, also known as Opdivo, MDX-1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in WO2006 / 121168 and U.S. Patent No. 8,008,449. The heavy and light chains of nivolumab are shown by the amino acid sequences illustrated in SEQ ID NOs: 25 and 26, respectively.

[0218] cemiplimab-rwlc, also known as cemiplimab, LIBTAYO, or REGN2810, is a recombinant human IgG4 monoclonal antibody described in WO2015112800 and U.S. Patent No. 9,987,500. The heavy and light chains of cemiplimab-rwlc are shown by the amino acid sequences shown in SEQ ID NOs: 101 and 102, respectively.

[0219] In a particular embodiment, the anti-PD-1 antibody comprises (i) a pembrolizumab containing three HC-CDRs fused to or linked to an effector-silencing HC constant domain. H (ii) V of three LC-CDRs containing pembrolizumab fused to or linked to the LC κ or λ constant domain. L The three HC-CDRs contain SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 respectively, and the three LC-CDRs contain SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 respectively.

[0220] In a particular embodiment, the anti-PD-1 antibody comprises (i) a nivolumab containing three HC-CDRs fused to or linked to an effector-silencing HC constant domain. H (ii) V of three LC-CDRs containing nivolumab fused to or linked to the LC κ or λ constant domain. L The three HC-CDRs contain SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 respectively, and the three LC-CDRs contain SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 2 respectively.

[0221] In a particular embodiment, the anti-PD-1 antibody comprises (i) a V-CDR of cemiplimab-rwlc comprising three HC-CDRs fused to or linked to an effector-silencing HC constant domain. H (ii) V of three LC-CDRs containing nivolumab fused to or linked to the LC κ or λ constant domain. L .

[0222] In a particular implementation, the anti-PD-1 antibody comprises (i) the V of pembrolizumab. H and V L Structural domain, where V H The structural domains and effector-silenced HC constant structural domains are fused or connected, and V L(ii) V of nivolumab H and V L Structural domain, where V H The structural domains and effector-silenced HC constant structural domains are fused or connected, and V L The structural domain is fused or connected with the LC κ or λ constant structural domain; or (iii) the V of cemiplimab-rwlc H and V L Structural domain, where V H The structural domains and effector-silenced HC constant structural domains are fused or connected, and V L The domain is fused with or connected to the LC κ or λ constant domain. Pembrolizumab V H The domain contains the amino acid sequence shown in SEQ ID NO:29, and V L The domain contains the amino acid sequence shown in SEQ ID NO:30. Nivolumab V H The domain contains the amino acid sequence shown in SEQ ID NO:23, and V L The domain contains the amino acid sequence shown in SEQ ID NO:24. cemiplimab-rwlc V H The domain contains the amino acid sequence shown in SEQ ID NO:99, and V L The domain contains the amino acid sequence shown in SEQ ID NO:100. In a particular embodiment, V H and V L The domain may further include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0223] In a specific implementation, anti-PD-1 antibody V H Domains can be merged or connected to a specific V that is not currently connected to. H The IgG1, IgG2, IgG3, or IgG4 HC constant domain, or a IgG1, IgG2, IgG3, or IgG4 HC constant domain that has been modified to include one or more mutations in the Fc domain that silence the resulting anti-PD-1 antibody effector.

[0224] In some embodiments, the HC constant domain is an isotype of IgG1, IgG2, IgG3, or IgG4, which is modified by replacing the codon of Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, such as Gln, to deprive the asparagine (Asn) residue at position 297 of the HC constant domain. N- Glycosylation. In a further embodiment, this is modified to lack at position 297. N - Glycosylated IgGs further include one or more additional mutations disclosed herein for eliminating detectable effector functions. In a particular embodiment, the HC constant domain is the human HC constant domain. In a particular embodiment, the molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0225] In a particular embodiment, the present invention provides an anti-PD-1 antibody comprising an IgG4 HC constant domain modified to have an S228P substitution and further comprising P239G, D265A, or D265A / N297G amino acid substitutions in addition to the S228P substitution, wherein the position is identified according to the Eu number. In the particular embodiment described above, the IgG4 HC constant domain is a human HC constant domain. In a particular embodiment, the molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0226] In another embodiment, the anti-PD-1 antibody may comprise a human IgG1 isotype, wherein the Fc domain of the HC constant domain has been modified to silence the effector by replacing amino acids at positions 233 to 236 of IgG1 with corresponding amino acids of human IgG2HC and replacing amino acids at positions 327, 330, and 331 with corresponding amino acids of human IgG4HC, wherein said positions are identified according to Eu numbering. In a particular embodiment, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0227] In another embodiment, the Fc domain of the IgG1, IgG2, IgG3, or IgG4 HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, and said polypeptide does not show measurable binding to FcγRIIIA, FcγRIIA, and FcγRI or shows reduced binding, as determined by Biacore assays, compared to polypeptides containing the wild-type IgG constant domain region. These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al., Nature 406, 267-273 (July 20, 2000). In a particular embodiment, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved mutations or non-conserved mutations.

[0228] Tables 19-27 provide specific exemplary anti-PD-1 antibodies that can be combined with anti-CTLA-4 antibodies as disclosed herein for use in therapy to treat individuals with cancer. The invention also provides antibodies and compositions shown in the tables, each composition comprising the antibody shown in the tables and a pharmaceutically acceptable carrier. All HC amino acid substitutions in Tables 19-27 are based on the Eu numbering scheme.

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] (e) Exemplary anti-PD-1 antibody fragment

[0236] Exemplary anti-PD-1 antibody fragments that can be used in combination therapies of the present invention include any anti-PD-1 antibody fragment that binds to PD-1 and inhibits PD-1 binding to PD-L1, and further include the following anti-PD-1 antibody fragments that bind to PD-1 and compositions comprising the following anti-PD-1 antibody fragments and pharmaceutically acceptable carriers.

[0237] In a particular embodiment, the antibody fragment is pembrolizumab V comprising the amino acid sequence shown in SEQ ID NO:29. H and pembrolizumab V having the amino acid sequence shown in SEQ ID NO:30 L Fv or scFv.

[0238] In a particular embodiment, the anti-PD-1 antibody fragment is pembrolizumab V comprising the amino acid sequence shown in SEQ ID NO:29. H and pembrolizumab V having the amino acid sequence shown in SEQ ID NO:30 H F(ab).

[0239] In a particular embodiment, the anti-PD-1 antibody fragment is pembrolizumab V comprising the amino acid sequence shown in SEQ ID NO:29. H And F(ab')2 of pembrolizumab VH having the amino acid sequence shown in SEQ ID NO:30.

[0240] In a particular embodiment, the anti-PD-1 antibody fragment is nivolumab V comprising the amino acid sequence shown in SEQ ID NO:23. H and nivolumab V having the amino acid sequence shown in SEQ ID NO:24 H Fv or scFv.

[0241] In a particular embodiment, the anti-PD-1 antibody fragment comprises nivolumab VH having the amino acid sequence shown in SEQ ID NO:23 and nivolumab V having the amino acid sequence shown in SEQ ID NO:24. H F(ab).

[0242] In a particular embodiment, the anti-PD-1 antibody fragment is nivolumab V comprising the amino acid sequence shown in SEQ ID NO:23. H and nivolumab V having the amino acid sequence shown in SEQ ID NO:24 H F(ab')2.

[0243] In a particular embodiment, the anti-PD-1 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD containing a variable domain (V) of camel-only heavy chain antibodies. HHHowever, the ISVD must not contain, as disclosed in International Patent Applications WO2008071447, WO2017087587 and WO2017087589, CDR1 containing the amino acid sequence THAMG (SEQ ID NO:73), CDR2 containing the amino acid sequences VITWSGGITTYADSVKG (SEQ ID NO:74) or VITWSGGITTYADSVKG (SEQ ID NO:75), or CDR2 containing the amino acid sequences DKHQSSWYDY (SEQ ID NO:76) or DKHQSSFYDY (SEQ ID NO:75). CDR3 (NO:77) and variants containing one, two, or three mutations in CDR3 as shown in WO2008071447, except that ISVDs containing said CDRs in embodiments in which one or more ISVDs are fused or linked to an effector silencing antibody constant domain or Fc domain, such as any effector silencing antibody constant domain or Fc domain disclosed herein, are not excluded by the conditions.

[0244] (f) Exemplary anti-PD-L1 antibody

[0245] Exemplary anti-PD-L1 antibodies that can be used in combination therapies of the present invention include any anti-PD-L1 antibody that inhibits PD-1 binding to PD-L1, and further include the following anti-PD-L1 antibodies and compositions comprising the following anti-PD-L1 antibodies and pharmaceutically acceptable carriers. In a particular embodiment, the anti-PD-L1 antibody is selected from atezolizumab, avermab, and devarulumab.

[0246] In a particular implementation, the anti-PD-L1 antibody comprises (i) atezolizumab V H and V L Structural domain, where V H The structural domain merges or connects with the HC constant structural domain or the effector silences the HC constant structural domain, and V L The domain is fused or connected to the LC κ or λ constant domain, (ii) the V of avermab H and V L Structural domain, where V H The structural domain merges or connects with the HC constant structural domain or the effector silences the HC constant structural domain, and V L The domain is fused or connected to the LC κ or λ constant domain, or (iii) the V of devarulmab H and V L Structural domain, where V H The structural domain merges or connects with the HC constant structural domain or the effector silences the HC constant structural domain, and V L The domain fuses with or connects to the LC κ or λ constant domain. Devalumab VH The domain contains the amino acid sequence shown in SEQ ID NO:103, and V L The domain contains the amino acid sequence shown in SEQ ID NO:104. Acimenbazine V H The domain contains the amino acid sequence shown in SEQ ID NO:105, and V L The domain contains the amino acid sequence shown in SEQ ID NO:106. Atezolizumab V H The domain contains the amino acid sequence shown in SEQ ID NO:107, and V L The domain contains the amino acid sequence shown in SEQ ID NO:108. In a particular embodiment, V H and V L The domain further includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0247] In a specific implementation, anti-PD-L1 antibody V H Domains can be merged or connected to a specific V that is not currently connected to. H The IgG1, IgG2, IgG3, or IgG4 HC constant domain, or a IgG1, IgG2, IgG3, or IgG4 HC constant domain that has been modified to include one or more mutations in the Fc domain that silence the resulting anti-PD-L1 antibody effector.

[0248] In some embodiments, the HC constant domain is an isotype of IgG1, IgG2, IgG3, or IgG4, which is modified by replacing the codon of Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, such as Gln, to deprive the asparagine (Asn) residue at position 297 of the HC constant domain. N - Glycosylation. On the other hand, the Ser codon can be replaced by the Pro codon, or the Thr codon can be replaced by any codon other than the Ser codon, such as N297A, N297G, or N297D. Alternatively, all three codons can be modified. In a further embodiment, this modification is used to eliminate the codon at position 297. N - Glycosylated IgGs further include one or more additional mutations disclosed herein for eliminating detectable effector functions. In a particular embodiment, the HC constant domain is the human HC constant domain. In a particular embodiment, the molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0249] In a particular embodiment, the present invention provides an anti-PD-L1 antibody comprising an IgG4 HC constant domain modified to have an S228P substitution and further comprising P239G, D265A, or D265A / N297G amino acid substitutions in addition to the S228P substitution, wherein the position is identified according to the Eu number. In the particular embodiment described above, the IgG4 HC constant domain is a human HC constant domain. In a particular embodiment, the molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conserved or non-conserved mutations.

[0250] In another embodiment, the anti-PD-L1 antibody may comprise a human IgG1 isotype, wherein the Fc domain of the HC constant domain has been modified to silence the effector by replacing amino acids at positions 233 to 236 of IgG1 with corresponding amino acids of human IgG2HC and replacing amino acids at positions 327, 330, and 331 with corresponding amino acids of human IgG4HC, wherein the positions are identified according to Eu numbering. In a particular embodiment, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the substitutions may be conserved or non-conserved mutations.

[0251] In another embodiment, the Fc domain of the IgG1, IgG2, IgG3, or IgG4 HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, and said polypeptide does not show measurable binding to FcγRIIIA, FcγRIIA, and FcγRI or shows reduced binding, as determined by Biacore assays, compared to polypeptides containing the wild-type IgG constant domain region. These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al., Nature 406, 267-273 (July 20, 2000). In a particular embodiment, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the substitutions may be conserved mutations or non-conserved mutations.

[0252] Tables 28-36 provide exemplary anti-PD-L1 antibodies that can be combined with anti-CTLA-4 antibodies as disclosed herein for use in a therapy to treat individuals with cancer. The invention also provides antibodies and compositions shown in the tables other than antibodies 25-9 and 31-8, each composition comprising an antibody shown in the tables other than antibodies 25-9 and 31-8 and a pharmaceutically acceptable carrier. All HC amino acid substitution positions in Tables 28-36 are according to the Eu numbering scheme.

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] (g) Exemplary anti-PD-L1 antibody fragment

[0260] Exemplary anti-PD-L1 antibody fragments that can be used in combination therapies of the present invention include any anti-PD-L1 antibody fragment that binds to PD-L1 and inhibits PD-L1 binding to PD-1, and further include the following anti-PD-L1 antibody fragments and compositions, each composition comprising the following anti-PD-L1 antibody fragments and a pharmaceutically acceptable carrier.

[0261] In a particular embodiment, the anti-PD-L1 antibody fragment is devalumab V comprising the amino acid sequence shown in SEQ ID NO:103. H and devalumab V having the amino acid sequence shown in SEQ ID NO:104 L Fv or scFv.

[0262] In a particular embodiment, the anti-PD-L1 antibody fragment is devalumab V comprising the amino acid sequence shown in SEQ ID NO:103. H and devalumab V having the amino acid sequence shown in SEQ ID NO:104 H F(ab).

[0263] In a particular embodiment, the anti-PD-L1 antibody fragment is devalumab V comprising the amino acid sequence shown in SEQ ID NO:103. HAnd F(ab')2 of devalumab VH having the amino acid sequence shown in SEQ ID NO:104.

[0264] In a particular embodiment, the anti-PD-L1 antibody fragment is avermab V comprising the amino acid sequence shown in SEQ ID NO:105. H and acimetidine V having the amino acid sequence shown in SEQ ID NO:106 H Fv or scFv.

[0265] In a particular embodiment, the anti-PD-L1 antibody fragment comprises avermab VH having the amino acid sequence shown in SEQ ID NO:105 and avermab V having the amino acid sequence shown in SEQ ID NO:106. H F(ab).

[0266] In a particular embodiment, the anti-PD-L1 antibody fragment is avermab V comprising the amino acid sequence shown in SEQ ID NO:105. H and acimetidine V having the amino acid sequence shown in SEQ ID NO:106 H F(ab')2.

[0267] In a particular embodiment, the anti-PD-L1 antibody fragment is atezolizumab V containing the amino acid sequence shown in SEQ ID NO:107. H and atezolizumab V having the amino acid sequence shown in SEQ ID NO:108 H Fv or scFv.

[0268] In a particular embodiment, the anti-PD-L1 antibody fragment comprises atezolizumab VH having the amino acid sequence shown in SEQ ID NO:107 and atezolizumab V having the amino acid sequence shown in SEQ ID NO:108. H F(ab).

[0269] In a particular embodiment, the anti-PD-L1 antibody fragment is atezolizumab V containing the amino acid sequence shown in SEQ ID NO:107. H and atezolizumab V having the amino acid sequence shown in SEQ ID NO:108 H F(ab')2.

[0270] In a particular embodiment, the anti-PD-L1 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD containing a variable domain (V) of camel-only heavy chain antibody. HHHowever, the ISVDs mentioned herein do not include the anti-PD-L1 ISVDs disclosed in International Application WO2008071447 having SEQ ID NO:394-399 therein or in WO2009030285, both of which are incorporated herein by reference. However, ISVDs fused to or linked with an effector silencing antibody constant domain or Fc domain, such as any effector silencing antibody constant domain or Fc domain disclosed herein, are not excluded by the aforementioned conditions.

[0271] (h) Exemplary combination therapy dosing regimen

[0272] This invention provides an anticancer therapy that combines the immunostimulatory effects of a PD-1 blocker with the antitumor effects of a CTLA-4 blocker, but without the dermatological or intestinal irAEs generally observed with CTLA-4 blockers when administered in combination with PD-1 blockers. A key feature of this invention is that the CTLA-4 blocker lacks measurable binding to one or more FcRs, as determined in a Biacore assay, or exhibits reduced binding to one or more FcRs compared to wild-type antibodies of the same isotype, as measured in a Biacore assay. Therefore, the CTLA-4 blocker does not exhibit measurable effector function or exhibits reduced effector function, enabling the effector-silencing CTLA-4 blocker to be used in combination with a PD-1 blocker at doses and durations of administration unavailable with CTLA-4 blockers exhibiting effector function. This feature distinguishes the CTLA-4 blocker of this invention from currently available CTLA-4 blockers.

[0273] In a typical dosing regimen of this invention, the CTLA-4 blocker and the PD-1 blocker may be administered simultaneously to an individual at separate doses and in different forms. Typically, the CTLA-4 blocker of this invention may be administered in combination with the PD-1 blocker at least at the same dose, dosing frequency, and duration of treatment currently approved by the US FDA for ipilimumab / nivolumab combination therapy for a specific indication. However, combination therapy is not limited to the specific indication approved by the US FDA, but may include any indication that may benefit from the combination therapy of this invention. The currently approved dose is 1 mg / kg of ipilimumab following administration of nivolumab at a dose of 3 mg / kg. This dose combination may then be repeated every three weeks for up to four doses, while the dose of nivolumab continues thereafter as needed every two weeks. However, in a further embodiment, the CTLA-4 blocker of this invention may be administered in combination therapy at a dose greater than 1 mg / kg, for example, at least 3 mg / kg. In a further embodiment, the dose may be at least 10 mg / kg, and in an even further embodiment, the dose may be from about 1 mg / kg to 10 mg / kg. In a particular embodiment, the CTLA-4 blocker of the present invention can be administered at the same dosing frequency and duration of treatment as in approved ipilimumab / nivolumab combination therapy. In a particular embodiment, the CTLA-4 blocker of the present invention can be administered at the same dosing frequency and duration of treatment as nivolumab in approved ipilimumab / nivolumab combination therapy.

[0274] In certain embodiments of the combination therapy, the CTLA-4 blocker is administered in a dose not based on individual weight. Thus, in certain embodiments, the CTLA-4 blocker may be administered at a dose of about 10 mg to 300 mg. In further embodiments, the dose is selected from 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg.

[0275] In the combination therapy of this invention, the PD-1 blocker can be administered at the same dose, frequency of administration, and duration of treatment as approved for PD-1 blockers in monotherapy for a specific indication. The dose of the CTLA-4 blocker can be as cited above, and the CTLA-4 blocker can be administered at the same frequency of administration and duration of treatment as cited above, or at the frequency of administration and duration of treatment for a specific PD-1 blocker paired with the CTLA-4 blocker.

[0276] The specific dosages of currently marketed PD-1 blockers vary among PD-1 blockers, so that in specific embodiments of the combination therapy of the present invention, the dosage, dosing frequency, and / or duration of treatment can be at least the same as those approved by the US FDA for a specific PD-1 blocker for a specific indication. For example, pembrolizumab is approved for use at a dose of 200 mg every three weeks as needed (2 mg / kg up to 200 mg every three weeks for pediatric individuals (2 years to 18 years of age) as needed); nivolumab is approved at a dose of 3 mg / kg every two weeks; cemiplimab-rwlc is approved for use at a dose of 350 mg every three weeks as needed; atezolizumab is approved for use at a dose of 1200 mg every three weeks as needed; acitumab is approved for use at a dose of 10 mg / kg or 800 mg every two weeks as needed; and devalumab is approved for use at a dose of 10 mg / kg every two weeks as needed.

[0277] In certain embodiments of the combination therapy, the PD-1 blocker is an anti-PD-1 antibody or an anti-PD-1 antibody fragment, which may be administered at doses of about 150 mg to about 250 mg, about 175 mg to about 250 mg, about 200 mg to about 250 mg, about 150 mg to about 240 mg, about 175 mg to about 240 mg, or about 200 mg to about 240 mg. In some embodiments, the dose of the anti-PD-1 antibody or its antigen-binding fragment is 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mg. In further embodiments, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered every three weeks as desired. In another embodiment of the combination therapy of the present invention, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered every six weeks at a dose greater than 250 mg, for example, about 400 mg, as desired.

[0278] In a specific embodiment of the combination therapy, the PD-1 blocker is an anti-PD-1 antibody or an anti-PD-1 antibody fragment, which may be administered at a dose of about 10 mg / kg to about 1200 mg. In a further embodiment, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered every two to three weeks as needed.

[0279] While PD-1 blockers can be administered at least at the doses, frequencies, and durations of treatment approved for PD-1 blockers in currently marketed monotherapies, the actual doses, frequencies, and durations of treatment for any particular combination of the invention may differ from those approved for PD-1 blocker monotherapies. Therefore, in specific embodiments of the combination therapies of the invention, the dose, frequency, and duration of treatment of any particular PD-1 blocker in the combination therapy will be determined by clinical trials conducted for the combination therapy.

[0280] In a specific embodiment of the combination therapy, the PD-1 blocker is nivolumab or an effector-silencing variant of nivolumab, administered intravenously to the individual at a dose of 3 mg / kg over 30 to 60 minutes every two to three weeks, as needed, wherein each dose of the CTLA-4 blocker administered intravenously after the administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of the PD-1 blocker. In a specific embodiment, nivolumab or an effector-silencing variant of nivolumab is administered intravenously to the individual at an initial dose of 3 mg / kg over 30 minutes, followed by intravenous administration of the CTLA-4 blocker over 30 minutes on the same day for four doses every three weeks, and then nivolumab is administered intravenously at a fixed dose of 240 mg over 30 minutes every two weeks or 480 mg over 30 minutes every four weeks.

[0281] In a particular implementation, the PD-1 blocker is pembrolizumab or an effector silencing variant of pembrolizumab, administered intravenously to adult individuals at a dose of 200 mg over 30 minutes every three weeks or to pediatric individuals at a dose of 2 mg / kg over 30 minutes every three weeks up to a maximum of about 200 mg, wherein each treatment is followed by a dose of CTLA-4 blocker, wherein each dose of CTLA-4 blocker administered intravenously after administration of PD-1 blocker lasts for the same duration of treatment as PD-1 blocker or for a duration shorter or longer than the duration of PD-1 blocker.

[0282] In a particular implementation, the PD-1 blocker is pembrolizumab or an effector silencing variant of pembrolizumab, administered intravenously to an adult individual at a dose of 400 mg over 30 minutes every six weeks, wherein each treatment is followed by a dose of CTLA-4 blocker, wherein each dose of CTLA-4 blocker administered intravenously after administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of treatment of the PD-1 blocker.

[0283] In a specific embodiment of the combination therapy, the PD-1 blocker is cemiplimab-rwlc or an effector-silencing variant of cemiplimab-rwlc, administered intravenously to the individual at a dose of 350 mg over 30 minutes every three weeks, as needed, wherein each dose of the CTLA-4 blocker administered intravenously after the administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of treatment of the PD-1 blocker. In a specific embodiment, cemiplimab-rwlc or an effector-silencing variant of cemiplimab-rwlc is administered intravenously to the individual at an initial dose of 350 mg over 30 minutes every three weeks, as needed, followed by administration of the CTLA-4 blocker over 30 minutes on the same day.

[0284] In a specific embodiment of the combination therapy, the PD-1 blocker is atezolizumab or an effector-silencing variant of atezolizumab, administered intravenously to the individual at a dose of 1200 mg over 60 minutes every three weeks, as needed, wherein each dose of the CTLA-4 blocker administered intravenously after the administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of treatment of the PD-1 blocker. In a specific embodiment, atezolizumab or an effector-silencing variant of atezolizumab is administered intravenously to the individual at an initial dose of 1200 mg over 60 minutes every three weeks, as needed, followed by the administration of the CTLA-4 blocker over 30 minutes on the same day.

[0285] In a specific embodiment of the combination therapy, the PD-1 blocker is avermab or an effector-silencing variant of avermab, administered intravenously to the individual at a dose of 10 mg / kg or 800 mg over 60 minutes every two weeks, as needed, wherein each dose of the CTLA-4 blocker administered intravenously after the administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of treatment of the PD-1 blocker. In a specific embodiment, avermab or an effector-silencing variant of avermab is administered intravenously to the individual at a starting dose of 10 mg / kg or 800 mg over 60 minutes every two weeks, as needed, followed by the administration of the CTLA-4 blocker over 30 minutes on the same day.

[0286] In a specific embodiment of the combination therapy, the PD-1 blocker is devalumab or an effector-silencing variant of devalumab, administered intravenously to the individual at a dose of 10 mg / kg over 60 minutes every two weeks, as needed, wherein each dose of the CTLA-4 blocker administered intravenously after the administration of the PD-1 blocker lasts for the same duration of treatment as the PD-1 blocker or for a duration shorter or longer than the duration of treatment of the PD-1 blocker. In a specific embodiment, devalumab or an effector-silencing variant of devalumab is administered intravenously to the individual at a starting dose of 10 mg / kg over 60 minutes every two weeks, as needed, followed by the administration of the CTLA-4 blocker over 30 minutes on the same day.

[0287] While currently approved CTLA-4 and PD-1 blockers are provided in formulations at concentrations allowing intravenous administration to an individual over a timeframe of 30 to 60 minutes, the combination therapy of the present invention is contemplated in embodiments where the CTLA-4 blocker and / or PD-1 blocker are each provided in formulations at concentrations allowing individual administration to an individual in a single injection. The ability to provide at least one of the two blockers in a single injection will significantly reduce the time required to administer both blockers to an individual.

[0288] In a further embodiment, the present invention provides a combination therapy in which a CTL-4 blocker and a PD-1 blocker are co-administered simultaneously. Co-administration can be achieved by providing the CTLA-4 and PD-1 blockers in separate formulations and simultaneously administering each formulation to the individual, either via separate IVs or by mixing the mixture before administering it to the individual via IV, or by injecting each formulation separately into the individual. Co-administration can also be achieved by providing the CTLA-4 and PD-1 blockers in a single formulation, which is then administered to the individual in a single IV or a single injection.

[0289] (i) Combination therapy

[0290] The combination therapy of the present invention can be used to treat any proliferative disease, particularly cancer. In specific embodiments, the combination therapy of the present invention can be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0291] In another embodiment, the combined treatment of the present invention can be used to treat pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancies.

[0292] Currently marketed PD-1 blockers are approved by the US FDA for the treatment of at least one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced stages), and squamous cell carcinoma of the skin. Therefore, the combination therapy of the present invention can be used to treat at least one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced stages), and squamous cell carcinoma of the skin.

[0293] (j) Combination therapy with chemotherapy

[0294] The combination therapy of the present invention can be administered in combination with chemotherapy to individuals with cancer. An individual may receive chemotherapy while undergoing the combination therapy of the present invention. An individual may receive the combination therapy of the present invention after completing chemotherapy. An individual may be given chemotherapy after the combination therapy has been completed. The combination therapy of the present invention can also be administered to individuals with recurrent or metastatic cancer who are undergoing or have completed chemotherapy, and who have experienced disease progression or recurrence.

[0295] Chemotherapy may include chemotherapeutic agents selected from the following:

[0296] (i) Alkylating agents, including but not limited to bifunctional alkylating agents, cyclophosphamide, nitrogen mustard, chlorambucil, and melphalan;

[0297] (ii) Monofunctional alkylating agents, including but not limited to dacarbazine, nitrosourea and temozolomide (oral dacarbazine);

[0298] (iii) Anthraquinones, including but not limited to daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and pentorubicin;

[0299] (iv) Cytoskeleton disruptors (taxanes), including but not limited to paclitaxel, docetaxel, abraxane, and tesotriene;

[0300] (v) Epomycins, including but not limited to ixaprone and utidelon;

[0301] (vi) Histone deacetylase inhibitors, including but not limited to vorinostat and romidesin;

[0302] (vii) Inhibitors of topoisomerase i, including but not limited to irinotecan and topotecan;

[0303] (viii) Inhibitors of topoisomerase II, including but not limited to etoposide, teniposide and tafluposide.

[0304] (ix) Kinase inhibitors, including but not limited to bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib.

[0305] (x) Nucleotide analogs and precursor analogs, including but not limited to, azacytidine, azathioprine, fluoropyrimidines (e.g., capecitabine, carmoflurane, doceflurane, fluorouracil, and nitrofluoridine), cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (prothioguanine).

[0306] (xi) Peptide antibiotics, including but not limited to bleomycin and actinomycin; platinum-based reagents, including but not limited to carboplatin, cisplatin and oxaliplatin;

[0307] (xii) Class retinols, including but not limited to retinoic acid, avitamin A, and bexarotine; and

[0308] (xiii) Vinca alkaloids and derivatives, including but not limited to vincristine, vinblastine, vincristine and vinblastine.

[0309] The dosage of a chemotherapy agent chosen for chemotherapy depends on several factors, including the serum or tissue turnover of the entity in the individual being treated, the level of symptoms, the immunogenicity of the entity, and the availability of target cells, tissues, or organs. The dosage of additional therapeutic agents should be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of administration of each additional therapeutic agent will depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the patient characteristics. Guidelines for selecting appropriate dosages of antibodies, cytokines, and small molecules are available. See, for example, Wawrzynczak (1996). Antibody Therapy , Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases , Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th edition); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Appropriate dosing regimens may be determined by the clinician, for example, using parameters or factors known or suspected in the art to affect or predict their influence on treatment, and will depend on, for example, the individual's clinical history (e.g., previous treatments), the type and stage of cancer to be treated, and biomarkers of response to one or more therapeutic agents in combination therapy.

[0310] For example, pembrolizumab is currently approved by the U.S. FDA for combination therapy for (i) the treatment of non-small cell lung cancer (NSCLC), including pembrolizumab with pemetrexed and platinum chemotherapy or carboplatin and paclitaxel or nanoparticle albumin-bound paclitaxel; and (ii) the treatment of head and neck squamous cell carcinoma (HNSCC), including pembrolizumab with platinum-based chemotherapy. Atezolizumab is currently approved for combination therapy for the treatment of NSCLC, including bevacizumab (an anti-VEGF-A antibody marketed under the brand name Avastin), paclitaxel, and carboplatin.

[0311] Therefore, the present invention contemplates embodiments of the combination therapy of the present invention that further include a chemotherapy step, including platinum-based chemotherapy, pemetrexed and platinum-based chemotherapy, or carboplatin and paclitaxel or nanoparticle albumin-bound paclitaxel. In certain embodiments, the combination therapy having a chemotherapy step can be used to treat at least NSCLC and HNSCC.

[0312] Further combination therapy with chemotherapy steps can be used to treat any proliferative disease, particularly cancer. In specific embodiments, the combination therapy of the present invention can be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0313] In another implementation, combination therapy, further combined with chemotherapy steps, can be used to treat pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancies.

[0314] In certain implementations, combination therapy with a chemotherapy step can be used to treat one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced), and squamous cell carcinoma of the skin.

[0315] The following examples are intended to facilitate a further understanding of the present invention.

[0316] Example 1

[0317] Inducing intestinal inflammatory irAEs requires Fc-function.

[0318] Intestinal inflammatory adverse events (irAEs) have been observed in cancer patients during immunotherapy with anti-CTLA-4 antibody monotherapy or in combination with anti-PD-1 antibodies. Preclinical studies in constitutive and conditional knockout models of CTLA-4-deficient mice have demonstrated the development of deeply immune-mediated inflammatory disease in multiple organs. However, treatment with alternative anti-CTLA-4 antibodies in syngeneic tumor models has not been reported to induce significant irAEs predicting the toxicity observed in cancer patients. Similarly, histopathological evaluation of intestinal inflammation was assessed in CT26 syngeneic or xenograph models (mice inoculated with the CT26 colon cancer cell line). Fc-active anti-mouse CTLA-4 mAb 9D9-mIgG was used. 2a CT26 tumor-bearing mice (q4x4) treated with α-CTLA-4 or α-CTLA4 resulted in minimal granulocytic infiltration (grade 1 out of 5) observed in the lamina propria intestinal tissue. Granulocytic infiltration was not observed in cohorts treated with Fc-mutant anti-mCTLA-4 mAb 9D9-mIgG1-D265A (α-CTLA-4 (D265A)), Fc-free anti-mCTLA-4 ISVD F894 (CTLA-4 Nab), or the allotype. However, no accompanying ulceration or other tissue damage was observed.

[0319] The α-CTLA-4 (D265A) Fc- mutant lacks measurable affinity for the Fcγ receptor (Nimmerjahn et al., Immunity, 23: 41-51 (2005)) and therefore lacks Fc-effector function. In mice, the antitumor efficacy of anti-mouse CTLA4 mAb monotherapy depends on the antibody's ability to mediate the elimination of regulatory T cells within the tumor via Fc-effector function (Selby et al., in the cited book). Therefore, it is not expected that both CTLA-4 Nab and α-CTLA-4 (D265A) will have monotherapy antitumor benefits.

[0320] Minimal histopathological findings prompted us to evaluate gene expression profiles as a potentially more sensitive means of detecting markers of intestinal inflammatory cell activation. We utilized our previously developed PCR gene expression experimental group (Cayatte, C et al.) for proteomic and profilomic analysis of genes associated with intestinal inflammation in fecal samples and biopsies from preclinical models and patients of inflammatory bowel disease (IBD). Clinical and Translational Gastroenterology , 3: e10(2012)).

[0321] Gene expression profiles were measured in small and colonic tissue samples at various time points after the start of α-CTLA-4 treatment and compared with α-CTLA-4 (D265A) to specifically assess the role of Fc-function in inducing intestinal inflammation. Figure 1A As illustrated in the examples, the expression of many intestinal inflammatory genes was upregulated in proximal small intestine samples from mice treated with α-CTLA-4, but not in mice treated with α-CTLA-4 (D265A). These results suggest that, in a subclinical context, the manifestation of intestinal inflammatory pathways can be detected through gene expression in small and colonic tissues.

[0322] Upregulation of genes associated with intestinal inflammation allows us to assess the impact of continued treatment on the progression of clinical enterocolitis, as observed in patients treated with ipilimumab after 6 to 7 weeks or longer of treatment (Samaan et al., Nat. Rev. Gastroenterol. Hepatol. 15: 222-234 (2018)). To assess the relative effects of CTLA-4 blockade and Fc-function on intestinal inflammation and progression to enterocolitis, BALB / c mice were administered α-CTLA-4, α-CTLA-4 (D265A), twice weekly. Two groups of mice were treated with Fc-active α-CTLA-4: one group with CT26 tumors and one group of tumor-free BALB / c mice used for the first time in experiments, to assess the potential contribution of tumor growth and tumor immunity induction to the induction of intestinal inflammation. Body weight and body condition scores were assessed twice weekly throughout the duration of continued treatment to monitor the progression of enterocolitis.

[0323] In mice treated with both the allotype antibody and α-CTLA-4 (D265A), body weight continued to increase until day 50. In contrast, mice treated with α-CTLA-4 showed a decrease in average body weight after approximately 35 to 40 days. Figure 1B As assessed in FITC-glucan-fed mice on days 49 and 50, intestinal permeability was increased in the α-CTLA-4 treatment group, but not in mice treated with α-CTLA-4 (D265A). Figure 1C Histological evidence of inflammation in the proximal small intestine and colon, as assessed by pathologists (LA), revealed progression to moderate to severe enterocolitis in the α-CTLA-4 treatment group. Figure 1D and Figure 1EIt exhibits widespread immune infiltration, mucosal thickening, and goblet cell loss. In contrast, no enterocolitis was observed in mice treated with α-CTLA-4 (D265A), providing evidence that Fc-function is required for CTLA-4 blockade-induced enterocolitis. Notably, tumor growth and antitumor responses are not required for α-CTLA-4-induced intestinal inflammation.

[0324] It has been previously reported that mouse IgG with high FcγR-affinity can be used to... 2a In experiments comparing chimeric antibodies with mutant IgG1 chimeras that lack detectable FcγR-binding, a strong antitumor response to monotherapy requires an anti-CTLA-4 antibody with strong FcγR function (Selby et al., Cancer Immunol. Res. 1: 32-42 (2013)). T in tumors reg The specific exclusion surface is the use of mouse IgG 2a (mIgG) 2a Anti-CTLA-4 antibodies on the main chain play a key role in the observed strong monotherapy (Simpson et al., J. Exp. Med. 210:1695-710 (2013). Recent reports demonstrating the importance of CD28 for anti-PD-1 efficacy (see References) suggest that enhancing CD28 function by blocking the stronger interaction of CTLA-4 with ligands CD80 and CD86 using CTLA-4 antagonists may be possible without the need for T reg In the absence of TILs, it provides strong combined antitumor efficacy. ISVDs specific to mouse CTLA-4 (mCTLA-4) that can inhibit binding to CD80 and CD86 are derived from single-domain V antibodies derived from heavy-chain camel-derived antibodies. HH Antibody fragments were developed. These small 15 kDa proteins lack the Fc region and therefore do not bind FcγRs. For comparison with antibodies that are fully Fc functional, we utilized α-CTLA-4 (see References), which has previously demonstrated strong monotherapy antitumor efficacy. To compare with the potential impact of binding different CTLA-4 epitopes, we utilized α-CTLA-4 (D265A), which lacks detectable affinity for FcγRs, to contrast with the need for strong FcγR-function for both antitumor efficacy and tolerability.

[0325] CTLA-4 nAb consists of two anti-CTLA-4 V connectors connected via a 35GS connector. HH The structural domain and anti-human albumin V as a half-life extension (HLE) subunit HHDomain composition, see SEQ ID NO: 61). Compared to α-CTLA-4 (100%) used as a reference antagonist, anti-CTLA-4 V HH The inhibition rate for CD80 and CD86 was 96%. Figure 2A (and Figure 2B). Regarding the ability to enhance proliferation ( Figure 2C ), IFNγ ( Figure 2D ) and IL-2 response ( Figure 2E In an MLR-based in vitro bioassay, CTLA-4 nAb was further compared with α-CTLA-4 and effector-silenced α-CTLA-4 (D265A).

[0326] PD-1 combined with immunotherapy does not require CTLA-4 blocking of Fc-function.

[0327] The relative antitumor efficacy induced by monotherapy with three CTLA-4 antagonists and combination therapy with the anti-mouse PD-1 antibody mDX400 (α-PD-1) was evaluated by measuring changes in tumor volume over time in an syngeneic CT26 colon cancer tumor model. Figures 3A-3E The syngeneic CT26 colon cancer tumor model showed only a moderate response to anti-PD-1 monotherapy. No antitumor activity was observed in the monotherapy cohort treated with CTLA-4 nAb or α-CTLA-4 (D265A), and compared with the isotype control (…). Figure 3A The difference is not significant (Group 1 in the text). Strong antitumor monotherapy activity was observed in mice treated with α-CTLA-4, consistent with previous reports demonstrating that the efficacy of antitumor monotherapy in mouse syngeneic tumor models requires Fc-function in the anti-CTLA-4 antibody (Simpson et al., J. Exp. Med. 210: 1695-710 (2013)). Figure 3A Group 2), and α-PD-1 alone provided low to moderate anti-tumor growth inhibition compared to α-CTLA-4. However, CTLA-4 nAb or combination therapy with α-CTLA-4 (D265A) and α-PD-1 provided a strong anti-tumor benefit not significantly different from that observed with α-CTLA-4 alone or in combination with α-PD-1. Figure 3A (Group 2). CTLA-4 nAb and α-CTLA-4 (D265A) bind to independent epitopes on CTLA-4, therefore the effects are not epitope-specific. Further evidence of combined benefits independent of Fc function enhancement is evident, given the similar antitumor responses observed in α-PD-1 combination therapy between CTLA-4 nAb, α-CTLA-4 (D265A), and α-CTLA-4. Figure 3DShowing Figure 3A The individual results of each treatment in 10 mice were summarized. Compared with mice receiving α-CTLA-4 (D265S) alone, CD8 T cell expansion and an increased CD8 / Treg ratio were observed in mice treated with α-CTLA-4 and mice receiving the combination of α-CTLA-4 (D265S) and α-PD-1. Figure 3B These results indicate that the combination of an Fc-deficient anti-CTLA-4 antagonist and an anti-PD-1 antagonist provides superior antitumor efficacy compared to monotherapy with an anti-PD-1 antagonist, and the combination provides antitumor efficacy similar to that of an Fc-functional anti-CTLA-4 antibody in monotherapy.

[0328] We investigated the regulation of immune response genes in tumors associated with effective cancer immunotherapy nine days after treatment initiation to elucidate potential complementary mechanisms related to strong binding activity. PCR-expression profile analysis of tumors from mice treated with α-CTLA-4 revealed strong upregulation of many genes associated with effective immunotherapy. Figure 3C This includes IFNγ, IFN-response genes, chemokines, pro-inflammatory cytokines, and MHC. Only modest upregulation was observed in tumors from mice treated with α-CTLA-4 (D265A), suggesting that the strong upregulation observed in α-CTLA-4-treated tumors is at least partially dependent on Fc-function. Moderate responses were also observed in tumors from mice treated with CTLA-4 nAb and α-PD-1. In contrast, enhanced upregulation of tumor immune response genes was observed in the cohort treated with either CTLA-4 nAb or α-CTLA-4 (D265A) plus α-PD-1. Figure 3D The results showed that neither CTLA-4 nAb nor α-CTLA-4 (D265A) exhibited antitumor activity in the absence of PD-1 blocking. These data support the hypothesis that the complementary mechanisms of pure CTLA-4 blocking and PD-1 blocking can provide a strong combined benefit in an Fc-independent manner.

[0329] Non-Fc-functional CTLA-4 blockade in combination with anti-PD-1 provides a superior therapeutic index.

[0330] A significant feature of immune restriction point blockade is the clinically validated combined benefit of anti-PD-1 and anti-CTLA-4 antibodies, leading to superior clinical efficacy compared to targeting either restriction point pathway alone. However, immune-related toxicities (irAEs) associated with combination therapy with anti-PD-1 and CTLA-4 blockade have been linked to increased induction of intestinal inflammation in patients (Ribas & Wolchok, Science 359: 1350-1355 (2018)). Furthermore, both α-CTLA (D265A) and Fc-free CTLA-4 nAb require combination with α-PD-1 to induce strong anti-tumor immunity. To compare with the potential impact of strong tumor immunity on the induction of intestinal inflammation, we examined the profiling of intestinal inflammation expression in mice treated with combination therapy of α-PD-1 plus α-CTLA, α-CTLA (D265A), or CTLA-4 nAb after 5 treatments, starting on day 18. Figure 4D ).

[0331] To assess the relative effects of CTLA-4 blockade and Fc-function on intestinal inflammation, BALB / c mice used for the first time in the experiment were administered twice weekly with α-CTLA, α-CTLA (D265A), Fc-free CTLA4 nAb, α-PD-1, or a combination of α-PD-1 and various anti-mCTLA4 antagonists. Body weight and body condition scores were assessed twice weekly throughout the study. Until approximately day 20, mice in all groups had gained body weight ( Figure 4A In mice treated with isoform, α-CTLA (D265A), CTLA4nAb, or α-PD-1, or combinations thereof, body weight continued to increase until day 50. Mice treated with α-CTLA showed a decrease in average body weight to near pre-treatment levels by day 50, approximately after day 30. From day 20 to day 50, combined administration of α-CTLA and α-PD-1 resulted in a more rapid decrease in average body weight to below pre-treatment levels. Notably, in mice treated with α-CTLA, 2 out of 8 mice showed a decrease in body condition score from day 42 onwards, and in mice treated with α-CTLA in combination with α-PD-1, the score decreased to 2 (under-conditioned) from day 28 onwards. These cohorts exhibited shiny, scraggly fur, and swollen abdomens were observed in these cohorts.

[0332] Inflammation analysis was performed 7 weeks after administration, at which time mice treated with α-CTLA showed weight loss over a period of time, which worsened when combined with α-PD-1 (Group B in Figure 4A). In contrast, neither the effector-silencing CTLA-4 blocker nor α-PD-1 showed any significant weight loss during the said time period compared to isotype controls. Figure 4B Group A in the middle.

[0333] All mice from the combination therapy group and four mice from the isotype control and α-PD-1 treatment groups were euthanized on day 50 for tissue collection. The remaining four mice from the isotype control and α-PD-1 treatment groups and all mice from the single-agent treatment groups were euthanized on day 54 for tissue collection. At necropsy, the proximal small intestine and colon were removed for RT-qPCR to determine the expression of inflammatory genes and to assess inflammation by histopathology.

[0334] A heatmap of gene expression in the proximal small intestine of each treatment group relative to the isotype control is shown in ( Figure 4D α-CTLA administration is sufficient to induce jejunal ( Figure 4D Upregulation of inflammatory genes in the colon (Fig. 4E). The combination of α-CTLA and α-PD-1 induced even stronger upregulation of inflammatory genes than α-CTLA monotherapy. In contrast, administration of CTLA-4nAb, when combined with α-PD-1, induced little or no intestinal inflammatory gene expression and only moderate upregulation. Similarly, administration of α-CTLA (D265A), alone or in combination with α-PD-1, resulted in minimal to low induction of inflammatory genes. Intestinal permeability, assessed in serum after FITC-dextran tube feeding, was significantly increased in mice treated with α-CTLA and mice receiving combination therapy with α-CTLA and α-PD-1.

[0335] The severity of inflammation in the proximal small intestine was scored by histological assessment of enteritis in the proximal jejunum at day 50. Histopathological evaluation showed that administration of α-CTLA induced mild to severe inflammation in most mice. In the cohort treated with a combination of α-CTLA and α-PD-1, continued treatment induced moderate to very severe inflammation in all mice. Figure 4B Mice with very severe enteritis faced jejunitis, diffuse neutrophilic lesions with a moderate number of mast cells, and degeneration of submucosal plexus neurons. In comparison, administration of CTLA-4 nAb or α-CTLA (D265A) did not induce inflammation on histopathological evaluation. Administration of CTLA-4 nAb in combination with α-PD-1 caused no inflammation or minimal to mild inflammation in a few mice. Administration of α-CTLA (D265A) in combination with α-PD-1 resulted in mild inflammation in only one of eight mice. Representative micrographs show the relative level of inflammation in each treatment group. Figure 4C ).

[0336] like Figures 5A-5C As shown, the α-CTLA-4 Fc effector function causes skin inflammation ( Figure 5A However, it is not a systemic inflammation, in which no detectable inflammation is found in the kidneys, liver, or lungs. Figure 5C The absolute numbers of IL-17-producing T cells, Foxp3+ Treg cells, and neutrophils in the ear skin were measured by flow cytometry. Figure 5B As shown, elevated levels of IL-17-producing T cells, Foxp3+ Treg cells, and neutrophils were present in ear skin from mice treated with α-CTLA-4 instead of α-CTLA-4 (D265A). These data collectively support the crucial role of Fc-effector function in inducing intestinal inflammation via anti-mouse CTLA-4 antibodies with effector function. In the BALB / c mouse model of enterocolitis, Fc-effector function contributes to anti-mouse α-CTLA-induced intestinal inflammation, while in mice treated with CTLA-4 nAb or α-CTLA (D265A), intestinal inflammation was mild or absent.

[0337] In summary, two properties are associated with the induction of intestinal inflammation by α-CTLA-4 in the CT26 tumor model. First, CTLA-4 specificity is required because Fc-functional isotype controls do not elicit the expression of inflammation-related genes. However, blocking the binding of CTLA-4 to the CD80 / CD86 ligand is insufficient to induce the upregulation of inflammatory genes in the intestines of mice treated with CTLA-4 nAb and α-CTLA-4 (D265A). Therefore, α-CTLA-4 is primarily associated with IgG... 2a The strong Fc-functional capacity of the isotype is essential for inducing intestinal inflammation.

[0338] The activation of intestinal inflammation is caused by T-independent processes. regs The exclusion of T eff Cell activation initiation

[0339] The mechanism of action of anti-CTLA-4 mediated anti-tumor immunity (MOA) is theoretically based on the regulation of T cells (T3). reg ) cells and their response to T effectors (T eff The pharmacokinetic (PD) effects of cell populations (CTLs, TH1 cells, etc.) are mediated. T cells within the tumor microenvironment... reg Cellular exclusion (TILs) is an important MOA for anti-CTLA-4 antibodies in murine syngeneic tumor models (Simpson et al., in the cited book). Furthermore, Fc-FcγR co-engagement of anti-CTLA-4 mAbs regulates T cell receptor (TCR) and CD28 signaling, thereby leading to T-independent... regExcluded enhanced T cell activation (Waight et al., Cancer Cell, 33: 1033-1047 (2018)).

[0340] To characterize the effect of α-CTLA-4 and CTLA-4 nAb on T regs Differential effects were observed in T effector cells, as measured by flow cytometry of tumor T cell populations (TILs), lamina propria of the colon, blood, and spleen 20 hours after subcutaneous administration. We were able to measure T cell counts from mice treated with α-CTLA-4 or α-CTLA-4 (D265A) using the anti-CTLA-4 mAb clone UC10-4B9 (ThermoFisher). regs The expression level of CTLA-4 in the spleen of CT26 tumor-bearing mice was observed to be low because they do not cross-block, thus enabling staining of drug-bound CTLA-4. As previously reported in the literature (Selby et al., in cited book; Simpson et al., in cited book), we observed CTLA-4 expression levels in the spleen of CT26 tumor-bearing mice (CTLA-4...). lo ) and tumor microenvironment ( Treg hi T in ) regs Differential expression in PBMCs. T regs CTLA-4 expressing bimodal levels lo-mid Interestingly, T from the lamina propria of the colon regs CTLA-4 expressing bimodal levels mid-hi CTLA-4 in the colon hi T regs CTLA-4 expression levels are similar to those in the TregTIL population. Due to its receptor density-dependent killing mechanism, differential CTLA-4 expression levels across various T cell populations affect the ability to eliminate cells under ADCC-mediated inhibition. Although T... reg The population typically expresses higher levels of CTLA-4, but T in the tumor setting... regs Expression levels were significantly higher than those found in the spleen (MFI = 2,400) (3.3-fold higher than the MFI = 8,100 in isotype controls of Treg TILs). Higher CTLA-4 levels were also observed. hi Model MFI = 10,000) from the lamina propria T of the colon regs For relative expression levels similar to T using flow cytometry reg TILs ( Figure 6A ).like Figures 6B-6D The example given is T regsSignificant exclusion was limited to TILs from the tumor microenvironment of mice treated with α-CTLA-4, which had the highest CTLA-4 expression density. Treatment with Fc-mutant α-CTLA-4 (D265A) lacking Fc-function did not lead to TILs. reg exclude.

[0341] Based solely on the assumption that cells expressing high CTLA-4 levels will tend to be excluded by α-CTLA, we predict CTLA-4 levels in the lamina propria. hi T regs Will be excluded, similar to T reg TIL population. Surprisingly, TILs were isolated solely from tumors in mice treated with α-CTLA-4. regs TILs appear to have been excluded. TILs derived from the lamina propria of the colon in mice treated with α-CTLA-4... regs It appears that this cannot be ruled out. No Ta derived from the lamina propria of the colon was observed in mice treated with α-CTLA-4. regs The detectable exclusion suggests that the induction of intestinal inflammation is not caused by T cells in the intestinal mucosa. regs The loss of activation.

[0342] We investigated T cells that may have been one of the causes of α-CTLA-4-induced intestinal inflammation, and their inhibitory function. regs Possible phenotypic changes in the lamina propria (LP) of the colon from FoxP3 GLD report mice implanted with MC38 tumors. regs Sorting is performed for use with T reg PCR expression profile analysis of function-related genes ( Figure 7A However, compared with mice treated with α-CTLA-4 (D265A), LPT from α-CTLA... reg No significant differences in gene expression were observed in the cells. Using CD45RB for colitis... hi The T-cell metastasis model further investigated the effect of Fc-function on T cells. regs The potential impact. regs Cells were infected with CD45RB hi Passive transfer of T cells protected mice from the development of colitis. Figures 7B-7C ). Treatment with α-CTLA co-administered T reg Cells and CD45RB hi T cells in mice lead to T reg Loss of protection and development of colitis. In comparison, no loss of protection was observed in mice treated with CTLA-4nAb. Figures 7B-7C ). Flow cytometry-sorted colonic Foxp3+ T cells from mice 24 hours after treatment.reg Cellular gene expression profiles showed a significant upregulation of gene expression in mice treated with α-CTLA-4 compared to cells treated with CTLA-4 nAb or isotype control, and also compared to cells treated with CD45RB. 高 Observed similarities. In summary, these results suggest that Fc-mediated T... reg Exclusion criteria are not essential for T cells that induce intestinal inflammation via α-CTLA-4, but are relevant in response to intestinal inflammation. regs The adjustment function may be adjusted.

[0343] The restricted expression of CTLA-4 and CD28 on T cells and CD80, CD86, and FcγR on antigen-presenting cells may be important for T-independent expression. reg The activation of Fc-functional α-CTLA-4 nAb in excluded T cells plays a key role (Waight et al., Cancer Cell , 33: 1033-1047, 2018). Enhanced Fc-activation in tumors is advantageous, but may contribute to unintended irAEs in intestinal tissue. We investigated the potential contribution of Fc-function to the activation of immune effector cells in intestinal tissue. Flow cytometry analysis of CD16 / 32 expression on macrophages from both tumor and colon cells showed significantly higher FcR levels on antigen-presenting cells compared to splenic macrophages ( ). Figure 8A & 8B). In addition, CD45 was found in tumors and the lamina propria of the colon. + CD11b + F4 / 80 + The proportion of macrophages was significantly higher in the spleen than in the spleen. Figure 8C Fc-function is required for the activation of IL1β, TNFα, and IFNγ cytokine responses in intestinal tissue and is evident as early as 10 days after the start of treatment, long before obvious signs of intestinal inflammation, thus suggesting a key role in irAE induction. Figure 8C ).

[0344] Cytokine responses in CD4 T cells isolated from the lamina propria of the colon of α-CTLA-4-treated mice one month after treatment showed a higher proportion of IL-17, TNFα, and IFNγ-producing cells associated with Fc-mediated intestinal inflammation. Figure 8E Furthermore, α-CTLA-4 treatment also induced a Fc-dependent increase in neutrophils ( ). Figure 8E In summary, the results indicate that CTLA-4 blockade-related intestinal inflammation is induced by Fc-mediated effector cell activation, enhanced by FcγR-antibody enhancement via cell bridging of APCs and T cells, leading to stimulation of inflammatory cytokine responses, such as... Figure 11 As illustrated in the example, Fc-mediated intestinal inflammation induction can be independent of Tregs, excluding induction by effector T cells.

[0345] discuss

[0346] Several recent reports have supported the crucial role of Fc-function in syngeneic mouse cancer immunotherapy models for CTLA-4 blocking monotherapy (International Patent Application WO 2014 / 089113; Selby et al., Cancer Immunol. Res. 32-42 (2013); Vargas et al., Cancer Cell, 33: 649-663 (2018)). Ingram et al. reported in Proc. Natl. Acad. Sci. USA 115: 3912-3917 (2018) that providing anti-CTLA-4 ISVDs with antitumor efficacy requires fusing the Fc domain into the ISVD. Indeed, we observed a similar lack of efficacy when treating tumors with CTLA-4 nAb or α-CTLA-4 (D265A) as monotherapy. Tumor-infiltrating T cells expressing higher levels of cell surface CTLA-4... regs Specific exclusion has been shown to contribute to tumor efficacy in tumor models (Simpson et al., J.Exp. Med., 210: 1695-1710 (2013); Selby et al., Cancer Immunol. Res 1: 32-42 (2013).

[0347] α-CTLA-4-induced enterocolitis is not associated with detectable T cells present in the lamina propria. reg Excluding related factors. Our work elucidates the potential of CTLA-4 blockade in anticancer therapy by demonstrating potent antitumor activity when combined with an anti-PD-1 antibody without inducing strong intestinal inflammation. Achieving the combined benefits of CTLA-4 and PD-1 blockade does not require Fc-function. Combination therapy containing CTLA-4 nAb or α-CTLA-4 (D265A) and α-PD-1 induced activation in tumors of genes with similar IFNγ-related immune responses induced by α-CTLA-4, which has strong Fc-function. In contrast, strong intestinal inflammation progressing to enterocolitis was primarily observed in mice treated with α-CTLA-4 and increased when combined with the anti-PD-1 antibody mDX400. These results suggest that simple blockade of CTLA-4, thereby promoting CD28 activation, is sufficient to increase the antitumor response of exhausted T cells when combined with PD-1 blockade.

[0348] A previous report by Kamphorst et al. in Science 355: 1423-1427 (2017) demonstrated that PD-1 blocking rescue of exhausted CD8 T cells requires TCR-activated CD28 co-stimulation. Furthermore, a sister report by Hui et al. in Science 355: 1428-1433 (2017) demonstrated that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation of Shp2 phosphatases recruited via PD-1, and that CD28 is preferentially dephosphorylated. Our results suggest that simply blocking CTLA-4-mediated complementary activation of the TCR co-receptor CD28 with a non-Fc-functional anti-CTLA-4 antibody and blocking PD-1-mediated CD28 dephosphorylation may be sufficient to achieve the combined benefits for cancer immunotherapy. The advantage of this simple combined blocking without Fc-mediated enhanced activation via Fc-FcγR bridging may be that it allows for a larger therapeutic index, enabling higher dose ranges and longer treatment durations. Due to the lower risk profile of intestinal inflammation-related adverse events (irAEs), these advantages can also facilitate further combination with standard chemotherapy treatments.

[0349] Experimental Procedure

[0350] mice

[0351] Wild-type C57BL / 6J mice were obtained from the Jackson Laboratory. Wild-type Balb / c and CB17-SCID mice were obtained from Taconic. B6.Foxp3GDL (GFP-DTR-luciferase) mice were produced and maintained under specific pathogen-free conditions and kept in air-filtered microisolators at the Merck Research Laboratories (MRL) animal facility in Palo Alto, California. All animal procedures were approved by the MRL Institutional Animal Care and Use Committee in accordance with the guidelines for Assessment and Accreditation of Laboratory Animal Care.

[0352] Tumor attack and treatment

[0353] For syngeneic tumor experiments, CT26, MC38, and MB49 tumor models were used. 8- to 12-week-old Balb / c or C57BL / 6J mice were injected subcutaneously (sc) with 3 x 10⁻⁶ saturates in the flank. 5CT26 cells were observed. Tumor diameter was measured using electronic calipers, and tumor volume was calculated as length × width × width × 1 / 2. When the tumor reached approximately 100 mm... 3 Treatment began at that time. Twice a week, 10 mg / kg of α-CTLA-4, α-CTLA-4 (D265A), mouse anti-IgG1-D265A antibody isotype control, and mouse anti-IgG were administered. 2a Mice were treated with antibody isotype control and α-PD-1 antibody subcutaneously (sc). Mice were also treated twice weekly with 30 mg / kg of CTLA-4nAb or ISVD control sc.

[0354] α-CTLA-4 comprises HC having the amino acid sequence shown in SEQ ID NO: 58 and LC having the amino acid sequence shown in SEQ ID NO: 59.

[0355] α-CTLA-4 (D265A) comprises HC having the amino acid sequence shown in SEQ ID NO: 60 and LC having the amino acid sequence shown in SEQ ID NO: 60.

[0356] α-PD-1 comprises HC having the amino acid sequence shown in SEQ ID NO: 63 and LC having the amino acid sequence shown in SEQ ID NO: 64.

[0357] CTLA-4 nAb contains the amino acid sequence shown in SEQ ID NO: 61.

[0358] Anti-PD-1 ISVD F037 (PD-1 nAb) contains the amino acid sequence shown in SEQ ID NO:62.

[0359] Induction of colitis and skin inflammation

[0360] For the first time used in the experiment, 8- to 12-week-old Balb / c mice were administered 20 mg / kg of α-CTLA-4, α-CTLA-4 (D265A), mouse anti-IgG1-D265A isotype control, and mouse anti-IgG twice weekly. 2a Allotype control mice were treated with antibody subcutaneous (sc) for eight weeks. Mice were treated twice weekly with 30 mg / kg of CTLA-4 nAb or ISVD control sc. On day 55, plasma was collected for ELISA and Luminex assays. Organs were collected and processed as follows: 1) paraffin-embedded tissue sections fixed in 10% neutral buffered formalin and stained with H&E for histopathological evaluation; 2) flash-frozen in liquid nitrogen for further RNA extraction; or 3) placed in HBSS for cell isolation.

[0361] T-cell-driven colitis

[0362] Spleen cells from Balb / c mice were treated with magnetic beads (STEM CELL Technologies) and CD4 was purified. TCRb+ CD4+ CD25- CD45RB 高 T cells (CD45RB) 高 T cells) and low TCRb+CD4+CD25+CD45RB (T cells) reg Cells were sorted using FACS Aria (BD). 3 x 10 cells were injected intravenously. 5 CD45RB 高 T cells and 1x10 5 T reg Cells. Mice were administered 350 μg α-CTLA-4 or an isotype control or 600 μg CTLA-4 nAb or ISVD control intraperitoneally twice weekly. Mice were monitored and weighed for seven weeks post-injection.

[0363] Intestinal permeability

[0364] Four hours before the fluorescence measurement of FITC in serum, mice were fed FITC-glucan (4 kDa, Sigma-Aldrich) via tube.

[0365] Separation of colonic lamina propria, skin and tumor cells

[0366] Colonic lamina propria cells were isolated as follows: Epithelial cells were first removed by incubating 0.5 cm sections of intestinal tissue at 37°C for 20 minutes in Hank's buffer solution containing 5 mM EDTA and 10 mM HEPES, and this incubation was repeated once more. The remaining tissue was cut into small fragments and then digested under the same conditions in HBSS1X medium containing 0.250 mg / mL LIBERASE (Roche), 30 U / mL DNase I (Sigma-Aldrich), and dispersant (DISPASE) (Corning). The resulting cell suspension was layered onto a 40% / 80% PERCOLL gradient and centrifuged at 600 g for 10 minutes; LP cells were recovered at the interface.

[0367] Ear skin was excised and cultured in HBSS 1X containing 0.250 mg / mL LIBERASE (Roche), 30 U / mL DNase I (Sigma-Aldrich), and dispersant (DISPASE) (Corning) for 90 minutes at 37°C. The cell suspension was filtered and washed twice with HBSS 1X buffer. Tumor excises were cultured in HBSS 1X containing 0.250 mg / mL LIBERASE (Roche), 30 U / mL DNase I (Sigma-Aldrich), and dispersant (DISPASE) (Corning) for 30 minutes at 37°C. The cell suspension was filtered and washed twice with HBSS 1X buffer.

[0368] Histological samples from the colon, ear skin, liver, lung, and kidney strips were fixed overnight in 10% neutral-buffered formalin, transferred to 70% ethanol, routinely processed, embedded in paraffin, sectioned at 4–5 μm, and stained with hematoxylin and eosin (H&E). Colonic disease severity was assessed by a pathologist in a blinded manner according to three criteria: Inflammation: When present, characterized by infiltration of abundant (60–70%) mononuclear cells (macrophages and lymphocytes) and 30–40% neutrophils and band neutrophils. The inflammation score included the severity of infiltration, glandular loss, erosion, lumens distension, presence of crypt abscesses, and epithelial cell degeneration. Inflammation was graded on a scale of 0–4, where 0 = negative; 1 = minimal; 2 = mild; 3 = moderate; and 4 = severe. Apoptosis: The prevalence of apoptotic bodies was scored on a scale of 0-3, where 0 = negative; 1 = low; 2 = moderate; and 3 = high. Regeneration: The regenerative changes assessed included the prevalence of mitotic figures in the upper third of the mucosa, nuclear density (nuclear crowding) within individual glandular structures, and the regularity of the surface epithelium. Apoptosis was scored on a scale of 0-3, where 0 = negative; 1 = low; 2 = moderate; and 3 = high.

[0369] Flow cytometry and antibodies

[0370] Cells were resuspended in PBS and stained on ice for 30 minutes in the dark with a fixable viability stain (BD Bioscience). Cells were then resuspended in staining buffer (FBS, BD Bioscience) and stained on ice for 30 minutes with a combination of various direct fluorescent dyes conjugated. For intracellular antigens, surface-stained cells were permeabilized, fixed on ice with a Foxp3 staining buffer (eBiosciences) for 30 minutes, and then stained with specific antibodies. Mouse antibodies: CD45 (30-F11), CD8a (53-6.7), CTLA-4 (UC10-4B9), CD11c (HL3), CD11b (M1 / 70), TCRβ (H57-597), TCRγδ (GL3), CD4 (RM4-5 or GK1.5), CD25 (PC61), CD45RB (16A), Ly6G (1A8), F4 / 80 (T45-2342), CD16 / 32 (2.4G2), IFNγ (XMG1.2), IL-17A (TC11-18H10), TNFα (MP6-XT22), Foxp3 (FJK-16s). All antibodies were purchased from BD Biosciences, Biolegend, or eBioscience. All samples were sampled using an LSR II flow cytometer (BD). The data was analyzed using the FLOWJO software (Tree Star).

[0371] When cytokine production was measured by flow cytometry, cells were stimulated with 500 ng / mL iomycin and 50 ng / mL PMA (Sigma-Aldrich). One hour later, Brefield's styracin A (BD Bioscience) was added for another two hours before staining.

[0372] Mouse allogeneic mixed leukocyte response (MLR) assay

[0373] 2.105 mouse C57B6 / J (8-12 weeks old, female) spleen T cells were isolated using the EASYSEP Mouse T Cell Isolation Kit (STEMCELL) and compared with 1 x 10 5 Balb / c mouse spleen cells irradiated (at 2000 rads) were co-cultured with indicated concentrations of α-CTLA-4, α-CTLA-4 (D265A), CTLA-4 nAb, or an isotype control. On day 3, the supernatant was collected and IL-2 and IFN-γ production was measured by ELISA according to the manufacturer's specifications (Meso Scale Discovery). Then […] 3Pulsed cells with 1 μCi of H-thymidine (1 μCi per well) for 6 h or 16–18 h. Harvest cells onto a glass fiber filter using a cell harvester. Count the cells through the filter using a MicroBeta plate counter (PerkinElmer Microbeta 2450) according to the manufacturer's instructions.

[0374] Total RNA was isolated from tissues and cells and subsequently expressed using the Fluidigm BIOMARK platform. analyze.

[0375] For real-time PCR analysis, total RNA was isolated using either of two methods. Organ samples were homogenized in RNA STAT-60 (Tel-Test Inc., Friendswood, TX) using a polytron homogenizer, and then RNA was extracted using MagMAX-96 (ThermoFisher Scientific, Waltham, MA) for the Microarrays Kit, following the manufacturer's instructions. For cell samples, RNA was isolated using the ARCTURUS PICOPURE RNA Isolation Kit, following the manufacturer's instructions (ThermoFisher Scientific, Waltham, MA).

[0376] Total RNA treated with DNase was reverse transcribed using QUANTITECT Reverse Transcription (Qiagen, Valencia, CA) according to the manufacturer's instructions. Primers were commercially available from ThermoFisher Scientific (Foster City, CA). At least 2 ng of cDNA was pre-amplified for gene specificity using the Fluidigm BIOMARK manufacturer's instructions (Fluidigm, Foster City). Real-time quantitative PCR was then performed on the Fluidigm BIOMARK using two unlabeled primers, each at 900 nM, and a 250 nM FAM-labeled probe (ThermoFisher Scientific, Foster City, CA), with UNG-containing TAQMAN Universal PCR Master Mix. Samples and primers were run on 48x48 or 96x96 arrays according to the manufacturer's instructions (Fluidigm, Foster City). Ubiquitin levels were measured in separate reactions and used to normalize the data using the ∆Ct method. (Using the average cycling threshold value of ubiquitin and the gene of interest for each sample, equation 1.8^(Ct ubiquitin minus Ct gene of interest) x 10) 4 (Used to obtain normalized values). Primer reference sequences are available upon request.

[0377] Statistical method

[0378] Two-tailed paired and unpaired t-tests were used to calculate the statistical significance of the remainder of the study. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical analysis was performed using GraphPad PRISM 7 software.

[0379] Example 2

[0380] The antitumor efficacy of CTLA-4 nAb was evaluated in a mouse syngeneic MB49 tumor model. MB49 cells were derived from a urethral epithelial carcinoma cell line derived from adult C57BL / 6 mice by exposing primary bladder epithelial cell extracellular implants to 7,12-dimethylbenzo[a]anthracene (DMBA) for 24 hours followed by long-term culture. Syngeneic mouse models of bladder cancer have been widely used for over 35 years.

[0381] MB49 mouse bladder cancer cells were subcutaneously (sc) implanted into 80 mice, and the animals were divided into five treatment groups, with 10 mice in each group. The median initial tumor volume reached 103 mm.3 Mice were subcutaneously injected every four days for a total of four doses. An unrelated control ISVD (30 mg / kg, batch number 01AQL) and a 5 mg / kg mIgG1 isotype control mAb (batch number 64AIS) were administered as treatment controls. Treatment consisted of 30 mg / kg CTLA-4 nAb, 10 mg / kg Fc-active α-CTLA-4 (D265A), 5 mg / kg α-PD-1, or a combination of a CTLA4 target and α-PD-1. Tumor growth was monitored for 21 days after treatment initiation.

[0382] Figure 9A Tumor volumes in individual animals for each treatment group are shown. For the reactive treatment group, complete response (CR) up to day 21 is presented. Figure 9B The mean tumor volume and standard error of the mean are shown for each treatment group (starting number n=10 / group). Tumor volumes of animals removed from the study due to large tumor volumes are progressed normally (in the mean) until the last measurement was taken for the treatment group. Figures 9A-9B This shows that, compared with the CT26 colon tumor model (see...), Figure 3A As in the MB49 bladder tumor model and the MC38 colon tumor model, combination therapy with Fc-free CTLA-4 nAb and α-PD-1 provided strong antitumor benefits independent of Fc function.

[0383] Example 3

[0384] The antitumor efficacy of CTLA-4 nAb was evaluated in a mouse syngeneic MC38 tumor model. MC38 mouse colon cancer cells were implanted into 80 mice, and the animals were assigned to five treatment groups, with 10 mice in each group. The median initial tumor volume reached 246 mm. 3 Mice were injected with SC every four days for a total of four doses. An unrelated control ISVD (30 mg / kg) and a 5 mg / kg mIgG1 isotype control mAb were administered as treatment controls. Treatments included 30 mg / kg CTLA-4 nAb, 10 mg / kg Fc-active α-CTLA-4 (D265A), 5 mg / kg α-PD-1, or a combination of a CTLA4 target and α-PD-1. Tumor growth was monitored for up to 23 days after treatment initiation.

[0385] Figure 10A Tumor volumes in individual animals for each treatment group are shown. For the reactive treatment group, complete response (CR) up to day 23 is presented. Figure 10BThe mean tumor volume and standard error of the mean are shown for each treatment group (starting number n=10 / group). Tumor volumes of animals removed from the study due to large tumor volumes are progressed normally (in the mean) until the last measurement was taken for the treatment group. Figures 10A-10B This shows that, compared with the CT26 colon tumor model (see...), Figure 3A As in the MB49 bladder tumor model and the MC38 colon tumor model, combination therapy with Fc-free CTLA-4 nAb and α-PD-1 provided strong antitumor benefits independent of Fc function.

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[0409] While the invention has been described herein with reference to illustrative embodiments, it should be understood that the invention is not limited thereto. Those skilled in the art and understanding of the teachings herein will recognize additional modifications and embodiments within its scope. Therefore, the invention is limited only by the appended claims.

Claims

1. The use of anti-CTLA-4 antibodies and anti-PD-1 antibodies in the preparation of drugs for the treatment of colon cancer or bladder cancer. The CTLA-4 antibody comprises (i) a heavy chain (HC) having an HC variable domain (VH) consisting of the amino acid sequence shown in SEQ ID NO:7 and an HC constant domain consisting of the amino acid sequences shown in SEQ ID NO:38, 39, 40, 41, 42, 43, and 44, and (ii) a light chain (LC) having an LC variable domain (VL) consisting of the amino acid sequence shown in SEQ ID NO:8 and an LC constant domain consisting of the amino acid sequence shown in SEQ ID NO:

57. The anti-PD-1 antibody comprises: an HC consisting of the amino acid sequence shown in SEQ ID NO:27 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:28.

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