Anticancer combination therapy including CTLA-4 and PD-1 blockers

Abstract

An anticancer combination therapy comprising a CTLA-4 blocker and a PD-1 blocker is disclosed. In particular, a 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 a fragment thereof.

Description

Technology Field

[0001] (1) Field of invention

[0002] The present invention relates to an anticancer combination therapy comprising a CTLA-4 blocker and a PD-1 blocker. In particular, the present invention relates to a combination therapy, wherein the CTLA-4 blocker is an anti-CTLA-4 antibody having a reduced or immeasurable 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. Background Technology

[0003] (2) Description of related technology

[0004] Tumor immunotherapy is presumed to play a more prominent role in the treatment of various cancer indications. Clinical successes utilizing antibody blockade of immune checkpoint inhibitory receptors expressed on T cells, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have energized notable advancements in cancer immunotherapy. Monotherapy-based blockade of CTLA-4 or PD-1 with monoclonal antibodies (mAbs) has yielded enhanced antitumor responses and beneficial clinical outcomes in controlled, randomized clinical trials.

[0005] The outstanding feature of immune checkpoint blockade for the treatment of various cancers is the clinically validated benefit of combination therapies containing anti-PD-1 and anti-CTLA-4 antibodies. As an increasing amount of clinical data is published, it is becoming evident that anti-PD-1 / CTLA-4 combination therapy can provide superior clinical efficacy compared to targeting the checkpoint pathway alone. However, immune-related adverse events (irAEs) associated with anti-CTLA-4 antibodies were significant in both monotherapy settings and combination therapies with anti-PD-1 antibodies. For example, ipilimumab, an anti-CTLA-4 antibody marketed by Bristol-Myers Squibb under the brand name Yervoy and the only anti-CTLA-4 antibody approved by the U.S. Food and Drug Administration (FDA), constitutes a black box warning due to its potential to induce severe and fatal immune-mediated adverse reactions, such as inflammation of the intestines, liver, skin, hormone-producing glands, and / or eyes. Ipilimumab is also approved for combination therapy with the anti-PD-1 antibody nivolumab (marketed as Opdivo by Bristol-Myers Squibb) for advanced renal cell carcinoma and certain colorectal cancers; however, due to the risk of significant irAEs, ipilimumab is administered at a low 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 secondary melanoma (refer to Yervoy package inserts and label (July 2018)).

[0006] Nivolumab and pembrolizumab, both FDA-approved anti-PD1 mAbs, are humanized anti-PD1 IgG4 kappa antibodies, disclosed in U.S. Patent No. 8,008,449 and U.S. Patent No. 8,354,509, respectively. The IgG4 isotype Fc domain is generally recognized as having little detectable effector function.

[0007] Ipilimumab is a human anti-CTLA-4 IgG1 kappa antibody, disclosed in U.S. Patent No. 6,984,720. The heavy chain (HC) constant domain of the IgG1 isotype possesses an Fc domain generally recognized as having 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 suggested that Fc effector function is required for the efficacy of anti-CTLA-4 antibodies. For example, the literature [Ingram et al., Proc. Natl. Acad. Sci. USA 115: 3912-3917 (2018)] describes an anti-CTLA-4 alpaca heavy chain-alone antibody fragment (V) lacking the heavy chain Fc domain and its associated effector function in a mouse model. HH Although it did not possess antitumor efficacy, it was suggested that antitumor efficacy could be restored to the molecule by fusing it to the mouse IgG2 double-chain Fc domain, which exhibits effector function; the literature [Selby et al., Cancer Immunol. Res. 1: 32-42 (2013)] suggested that anti-CTLA-4 antibodies fused to or linked to an Fc domain mutated to eliminate effector function in a mouse model did not exhibit any antitumor activity. Also, refer to the literature [Simpson et al., J. Exp. Med.;210:1695-710 (2013)] and international patent application number WO2014089113.

[0008] Tremelimumab is a human anti-CTLA-4 human IgG2 antibody, disclosed in U.S. Patent No. 8,491,895. The human IgG2 isotype was selected to minimize potential effector function activity and thereby potentially reduce irAE. However, as presented in the literature [Vargas et al., Cancer Cell 33: 649-663 (2018)], tremelimumab maintains effector function; and the literature [Bertrand et al., BMC Med. 13: 211-214 (2015)] suggested that although tremelimumab can be administered at higher doses than ipilimumab, it can still induce irAE, particularly intestinal and cutaneous inflammatory immune-mediated toxicity. Also refer to the literature [Ribas et al., The Oncologist 12: 873-993 (2007)], [Schneider-Merck et al., J. Immunol. 184: 512-520 (2010)] and [Koenitzer et al., PLoS One. 10:e0145633 (2015)], which showed other human IgG2 antibodies that induce ADCC and ADCP equivalent to human IgG1 isoforms in vitro.

[0009] Another attempt to reduce irAEs with ipilimumab includes BMS-986249, a probody composed of ipilimumab linked to a proprietary shielding peptide that covers the active antigen-binding site of the antibody via a protease-cleavable linker. The shielding 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, literature [Pai et al., J. Clin. Invest. 129: 349-363 (2019)], and [Korman et al., Abstract SY09-01, AACR Annual Meeting Vol 77, issue 13 (2017)]).

[0010] The therapeutic efficacy of anti-CTLA-4 antibodies such as ipilimumab is regulatory T cells (T reg In light of studies suggesting that depletion of ) may be involved, it has been suggested that anti-CTLA-4 antibodies with enhanced ADCC activity, such as ipilimumab, would provide more effective antitumor activity than current antibodies. U.S. Patent No. 10,196,445 discloses several ipilimumab variants with enhanced ADCC activity.

[0011] Standard treatments for some anticancer regimens involve administering anti-PD-1 antibodies in combination with chemotherapy. While the antitumor 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 regimen may instead exacerbate gastrointestinal toxicity.

[0012] Clearly, anti-CTLA-4 antibodies that enable dosing at higher and more optimal levels without associated irAEs, particularly the skin and intestinal inflammatory immune-mediated toxicity currently associated with anti-CTLA-4 antibodies, are likely to allow for more effective therapy when combined with anti-PD-1 antagonists and optionally chemotherapy.

[0013] The inventors have discovered that certain CTLA-4 blockers that bind to CTLA-4 have reduced or immeasurable antitumor activity when administered as monotherapy, but can exhibit clinically relevant antitumor activity when used in combination therapy with PD-1 blockers. The inventors have also discovered that these specific CTLA-4 blockers can exhibit antitumor activity in CTLA-4 / PD-1 blockade combination therapy without inducing immune-mediated adverse events (irAEs) (including irAEs in the skin and intestine) associated with currently approved CTLA-4 / PD-1 blockade combination therapies. The CTLA-4 / PD-1 blockade combinations disclosed herein enable therapies with an increased therapeutic index compared to current CTLA-4 / PD-1 blockade combination therapies, including combination therapies including chemotherapy, which can lead to more effective cancer treatment with improved patient outcomes.

[0014] A specific CTLA-4 blocker used as part of the CTLA-4 / PD-1 blocking combination therapy of the present invention may be selected from the group consisting of (i) an effector-silencing anti-CTLA-4 antibody and (ii) an effector-silencing anti-CTLA-4 antibody fragment having an Fc domain comprising a deletion of a region within the Fc domain that binds to an Fc receptor (FcR) or a fragment crystallizable (Fc) domain. The effector-silencing antibody or antibody fragment exhibits (i) unmeasurable binding to one or more FcRs, as measurable in a Biacore assay, where an association constant in the micromolar range indicates unmeasurable binding, or (ii) measurable binding to one or more FcRs, as measurable in a Biacore assay, which is reduced compared to typical binding for antibodies of the same isotype. These specific CTLA-4 blockers are effector-silencing CTLA-4 blockers.

[0015] In certain embodiments, 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 without exhibiting irAEs, particularly cutaneous or intestinal inflammatory immune-related toxicity, typically associated with CTLA-4 / PD-1 blockade combination therapy.

[0016] The effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment disclosed herein may be used in combination with PD-1 or PD-L1 blockers at higher doses and for longer periods without exhibiting irAEs, particularly cutaneous or intestinal inflammatory immune-related toxicities, typically associated with CTLA-4 / PD-1 blockade combination therapy. Currently, the dose of the anti-CTLA-4 antibody ipilimumab approved for use in anti-CTLA-4 / PD-1 blockade combination therapy is 1 mg / kg compared to the 3 mg / kg or 10 mg / kg doses approved for use in monotherapy (see package inserts and labels for Yervoy (July 2018)) or the fixed dose of 100 mg or less considered for CTLA-4 / PD-1 blockade combination therapy in international patent application WO2018183408. Therefore, the CTLA-4 / PD-1 blockade combination therapy of the present invention may use an effector-silenced anti-CTLA-4 antibody or an effector-silenced anti-CTLA-4 antibody fragment at doses equal to or higher than the doses currently approved for anti-CTLA-4 antibodies in monotherapy. The effector-silenced anti-CTLA-4 antibody or anti-CTLA-4 antibody fragment disclosed herein may also be used in combination with an anti-PD-1 or anti-PD-L1 antibody at doses similar to those currently used or considered in CTLA-4 / PD-1 blockade combination therapy, but for a longer duration than currently available for anti-CTLA-4 antibodies, without exhibiting irAEs, particularly cutaneous or intestinal inflammatory immune-related toxicities, typically associated with CTLA-4 / PD-1 blockade combination therapy.

[0017] Accordingly, the present invention provides a combination therapy for treating cancer in an individual requiring cancer treatment, the method comprising administering (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 an individual having cancer to treat the cancer, wherein the effector-silencing CTLA-4 blocker exhibits antitumor activity in the combination therapy that is not present when administered to the individual in monotherapy without a PD-1 or PD-L1 blocker. In additional embodiments, the combination therapy does not induce or has a reduced risk of inducing immune-mediated adverse events (irAEs) in the intestine or skin during a course of the combination therapy greater than Grade 2 as defined in the Common Criteria for Adverse Events (CTCAE) version 5.0, compared to a combination therapy containing an anti-CTLA-4 antibody exhibiting effector function. In specific embodiments, the effector-silencing CTLA-4 blocker used as part of the combination therapy does not induce irAEs in the skin or intestine greater than Grade 2 during at least the first 10 weeks of the combination therapy. In a specific embodiment, the combination therapy does not result in a detectable irAE during at least the first 4 weeks of the combination therapy or an irAE greater than grade 1 during at least the first 4 weeks of the combination therapy.

[0018] In one embodiment, 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.

[0019] 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 specific 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 a region of the Fc domain that binds to the Fc domain or one or more FcRs, which silences the antibody fragment effector.

[0020] The present invention further provides an anticancer combination therapy comprising administering to an individual requiring cancer therapy (i) a PD-1 blocker selected from the group consisting of an anti-PD-1 antibody having an IgG4 or IgG2Fc domain, an effector-silenced anti-PD-1 antibody, and an effector-silenced anti-PD-1 antibody fragment; and (ii) a second formulation comprising an effector-silenced CTLA-4 blocker selected from the group consisting of an effector-silenced anti-CTLA-4 antibody and an effector-silenced anti-CTLA-4 antibody fragment.

[0021] The present invention further provides an anticancer combination therapy comprising administering to an individual requiring cancer therapy a formulation comprising: (i) a PD-1 blocker selected from the group consisting of an anti-PD-1 antibody having an IgG4 or IgG2Fc domain, an effector-silenced anti-PD-1 antibody, and an effector-silenced anti-PD-1 antibody fragment; and (ii) an effector-silenced CTLA-4 blocker selected from the group consisting of an effector-silenced anti-CTLA-4 antibody and an effector-silenced anti-CTLA-4 antibody fragment.

[0022] The present invention further provides an anticancer combination therapy comprising administering to an individual requiring cancer therapy (i) a PD-L1 blocker selected from the group consisting of an anti-PD-L1 antibody having an IgG4 or IgG2Fc domain, an effector-silenced anti-PD-L1 antibody, and an effector-silenced anti-PD-L1 antibody fragment; and (ii) a second formulation comprising an effector-silenced CTLA-4 blocker selected from the group consisting of an effector-silenced anti-CTLA-4 antibody and an effector-silenced anti-CTLA-4 antibody fragment.

[0023] The present invention further provides an anticancer combination therapy comprising administering to an individual requiring cancer therapy a formulation comprising: (i) a PD-L1 blocker selected from the group consisting of an anti-PD-L1 antibody having an IgG4 or IgG2Fc domain, an effector-silenced anti-PD-L1 antibody, and an effector-silenced anti-PD-L1 antibody fragment; and (ii) an effector-silenced CTLA-4 blocker selected from the group consisting of an effector-silenced anti-CTLA-4 antibody and an effector-silenced anti-CTLA-4 antibody fragment.

[0024] In a more specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or an additional amino acid substitution, insertion, and / or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; (ii) an amino acid substitution mutation selected from the group consisting of 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 a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and an amino acid substitution mutation selected from the group consisting of 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, It comprises an IgG1Fc domain having a mutated Fc domain that additionally includes insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0025] In a specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an amino acid substitution mutation selected from the group consisting of a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or an IgG2Fc domain having a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0026] In a specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody has an S228P amino acid substitution and comprises: (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an IgG4Fc domain further comprising a mutant Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0027] In a further embodiment of the combination therapy, the effector-silencing anti-CTLA-4 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, or comprises the same.

[0028] In a specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody or 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 the group consisting of 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.

[0029] In a specific embodiment of the combination therapy, the effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment is V of ipilimumab H and V L , V of tremelimumab H and V L , REGN4659's V H and V L , V of AGEN1884w H and V L , V of 8D2 / 8D2 (RE) H and V L , VH and VL of 8D2 / 8D2 (RE)-variant 1, V of 8D2H1L1 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 L , V of 8D3H3L3 H and V L , V of 8D2H2L15 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 Includes

[0030] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing the amino acid sequence presented in SEQ ID NO: 7 H and V containing the amino acid sequence presented in Sequence Identification Number: 8 L ; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 15 H and V containing the amino acid sequence presented in Sequence Identification Number: 16 L ; (iii) V containing the amino acid sequence presented in sequence identification number: 95 H and V containing the amino acid sequence presented in Sequence Identification Number: 96 L ; or (iv) V having the amino acid sequence presented in sequence identification number: 97 H and V having the amino acid sequence presented in Sequence Identification Number: 98 L Includes

[0031] In a specific embodiment, the effector-silencing anti-CTLA4 antibody or effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing the amino acid sequence presented in Sequence Identification No.: 73 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 74 L Domain; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 75 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 76 L Domain; (iii) V containing the amino acid sequence presented in sequence identification number: 77 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 78 L Domain; (iv) V containing the amino acid sequence presented in sequence identification number: 79 HV containing the amino acid sequence presented in Domain and Sequence Identification Number: 80 L Domain; (v) V containing the amino acid sequence presented in Sequence Identification Number: 81 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 82 L Domain; (vi) V containing the amino acid sequence presented in sequence identification number: 83 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 84 L Domain; (vii) V containing the amino acid sequence presented in sequence identification number: 85 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 86 L Domain; (viii) V containing the amino acid sequence presented in sequence identification number: 87 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 88 L Domain; (ix) V containing the amino acid sequence presented in sequence identification number: 89 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 90 L Domain; (x) V containing the amino acid sequence presented in Sequence Identification Number: 91 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 92 L Domain; or (xi) V containing the amino acid sequence presented in sequence identification number: 93 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 94 L Includes the domain.

[0032] 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.

[0033] In a further embodiment of the combination therapy, the effector-silencing CTLA-4 conjugate is an effector-silencing anti-CTLA-4 antibody fragment comprising one or more immunoglobulin monovariable domains (ISVDs), and each ISVD is a variable domain (V) of a camel and heavy chain monoantibody. HH V having ) but not including ); provided that none of the ISVDs include CDR1 containing the amino sequence FYGMG (Sequence ID: 69), CDR2 containing the amino acid sequence DIRTSAGRTTYADSVKG (Sequence ID: 70), and CDR3 containing the amino acid EMSGISGWDY (Sequence ID: 71) or EPSGISGWDY (Sequence ID: 72). HH (The above ISVD is as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087588), or V containing 1, 2, or 3 mutations in CDR3 as disclosed in WO2008071447 HH It is excluded that the ISVD comprising the CDR in an embodiment that does not include, and in which one or more ISVDs are fused to or linked to an effector-silencing heterologous HC domain or Fc domain comprising, for example, any one of the effector-silencing antibody HC domain or Fc domain disclosed herein, is not excluded by this proviso.

[0034] 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 semiplimab-rwlc. In a specific embodiment of the combination therapy, the anti-PD-1 antibody comprises (i) V of pembrolizumab H and V L ; (ii) V of nivolumab H and V L ; or (iii) V of semiplimab-rwlc H and V L Includes

[0035] In a further embodiment, the anti-PD-1 antibody or anti-PD-1 antibody fragment is (i) V having the amino acid sequence presented in Sequence Identification No.: 29 H and V having the amino acid sequence presented in Sequence Identification Number: 30 L ; (ii) V having the amino acid sequence presented in Sequence Identification Number: 23 H and V having the amino acid sequence presented in Sequence Identification Number: 24 L ; or (iii) V having the amino acid sequence presented in sequence identification number: 99 H and V having the amino acid sequence presented in Sequence Identification Number: 100 L Includes. In additional embodiments, the anti-PD1 antibody comprises (i) an HC having the amino acid sequence presented in sequence identification number: 27 and an LC having the amino acid sequence presented in sequence identification number: 28; (ii) an HC having the amino acid sequence presented in sequence identification number: 25 and an LC having the amino acid sequence presented in sequence identification number: 26; or (iii) an HC having the amino acid sequence presented in sequence identification number: 101 and an LC having the amino acid sequence presented in sequence identification number: 102.

[0036] In a specific embodiment of the combination therapy, the anti-PD-L1 antibody or anti-PD-L1 antibody fragment is (i) V of atezolizumab H and V L (ii) Avelumab's V domain; (ii) Avelumab's V H and V L Domain; or (iii) V of durvalumab H and V L Includes the domain.

[0037] In a further embodiment; the anti-PD-L1 antibody or anti-PD-L1 antibody fragment comprises (i) V having the amino acid sequence presented in sequence identification number: 103 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 104 LDomain; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 105 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 106 L Domain; or (iii) V comprising the amino acid sequence presented in sequence identification number: 107 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 108 L Includes the domain.

[0038] In a specific embodiment of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody may comprise an IgG1, IgG2, or IgG4Fc domain as disclosed herein, which may comprise C-terminal lysine or lack C-terminal lysine or C-terminal glycine-lysine dipeptide.

[0039] In a specific embodiment of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody comprises (i) an IgG2 or IgG4Fc domain; (ii) an IgG1, IgG2, or IgG4Fc domain comprising a mutation in the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iii) an IgG1Fc domain 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 a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iv) an IgG2Fc domain comprising N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S amino acid substitutions or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (v) an IgG4Fc domain comprising an S228P amino acid substitution and an N267A, P329G, or D265A / N297A amino acid substitution, or a mutated Fc domain further comprising 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.

[0040] In a specific embodiment 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.

[0041] In a further embodiment of the combination therapy, the anti-PD-1 antibody fragment or the anti-PD-L1 antibody fragment (each of which lacks an Fc domain) is a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), or an antigen-binding fragment dimer F(ab')2.

[0042] In a further embodiment of the combination therapy, the anti-PD-1 or anti-PD-L1 antibody fragment comprises one or more ISVDs, and each ISVD is a V of a camel-derived heavy chain monoclonal antibody. HH Includes

[0043] In a specific embodiment of the combination therapy, a CTLA-4 blocker is administered at a dose comprising about 1 mg / kg to about 3 mg / kg of CTLA-4 blocker or at a fixed dose of more than about 100 mg of CTLA-4 blocker regardless of the individual's body weight.

[0044] In a specific embodiment of the combination therapy, the CTLA-4 blocker is administered at a dose comprising 1 mg / kg to 3 mg / kg of CTLA-4 blocker.

[0045] In a specific embodiment of the combination therapy, the CTLA-4 blocker is administered at a dose comprising 3 mg / kg to 10 mg / kg of CTLA-4 blocker.

[0046] In a specific embodiment of the combination therapy, the CTLA-4 blocker is administered at a dose containing more than about 10 mg / kg of CTLA-4 blocker.

[0047] In a specific embodiment of the 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 body weight.

[0048] In a specific embodiment of the combination therapy, a PD-1 blocker is administered at a dose of 200 mg to 400 mg that does not depend on the body weight of the individual.

[0049] In a specific embodiment of the combination therapy, a PD-1 blocker is administered at a dose of 400 mg, regardless of the individual's body weight.

[0050] In a specific embodiment of the combination therapy, a PD-1 blocker is administered to the individual first and a CTLA-4 blocker is administered to the individual second, or a CTLA-4 blocker is administered to the individual first and a PD-1 blocker is administered to the individual second. In a specific embodiment, the PD-1 blocker and the CTLA-4 blocker are administered jointly.

[0051] In a specific embodiment of combination therapy, the individual is administered a chemotherapy agent before, in conjunction with, or after the combination therapy. In specific embodiments, the chemotherapeutic agent is actinomycin, total-trans retinoic acid, alitretinoin, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, carmofur, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotillon, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, ixabepilone, irinotecan, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexid, romidepsin, tegafur, It is selected from the group consisting of temozolomide (oral dacarbazine), tenifoside, thioguanine, topotecan, utidelon, valubicin, vemurafenib, vinblastine, vincristine, vindecin, vinorelbine, and vorinostat.

[0052] In a specific embodiment of the 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 lymphoma, kidney cancer, Hodgkin 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.

[0053] In a specific embodiment of the combination therapy, the cancer is pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0054] In a specific embodiment of the combination therapy, the individual is human, the CTLA-4 blocker binds to human CTLA-4, the PD-1 blocker binds to human PD-1, and the PD-L1 blocker binds to human PD-L1.

[0055] Antibodies and compositions

[0056] The present invention is each V H and V L Further providing an effector-silencing anti-CTLA-4 antibody or an effector-silencing anti-CTLA-4 antibody fragment comprising, wherein V H contains 3 heavy chain CDRs, and V L It contains three light chain CDRs, which bind together to CTLA-4. In a specific embodiment, CTLA-4 is human CTLA-4.

[0057] In a more specific embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions (provided that the effector-silencing anti-CTLA-4 antibody does not comprise ipilimumab consisting solely of the N297A substitution); (ii) an amino acid substitution mutation selected from the group consisting of 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 a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions;or (iii) a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and an amino acid substitution mutation selected from the group consisting of 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, It comprises an IgG1Fc domain having a mutated Fc domain that further includes insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0058] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or an additional amino acid substitution, insertion, and / or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; (ii) an amino acid substitution mutation selected from the group consisting of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an amino acid substitution mutation selected from the group consisting of a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or an IgG2Fc domain having a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0059] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody has an S228P amino acid substitution and comprises: (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an IgG4Fc domain further comprising a mutant Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0060] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody or 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 the group consisting of ipilimumab, tremelimumab, REGN4659, AGEN1884w, 8D2 / 8D2 (RE), 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15 and 8D2H2L17.

[0061] In certain embodiments, the effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment is V of ipilimumab H and V L , V of tremelimumab H and V L , REGN4659's V H and V L , V of AGEN1884w H and V L , V of 8D2 / 8D2 (RE) H and V L , V of 8D2H1L1 H and V L , V of 8D2H2L2 H and V L , V of 8D3H3L3 H and V L , V of 8D2H2L15 H and V L , or V of 8D2H2L17 H and V L Includes

[0062] In certain embodiments, the effector-silencing anti-CTLA-4 antibody or effector-silencing anti-CTLA-4 antibody fragment is 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 Includes. These variants are V H It includes the substitution of isoleucine for methionine at position 18 of the amino acid sequence.

[0063] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody or anti-effector-silencing CTLA-4 antibody fragment is (i) V having the amino acid sequence presented in Sequence Identification No. 7H and V having the amino acid sequence presented in Sequence Identification Number: 8 L ; (ii) V having the amino acid sequence presented in Sequence Identification Number: 15 H and V having the amino acid sequence presented in Sequence Identification Number: 16 L ; (iii) V having the amino acid sequence presented in sequence identification number: 95 H and V having the amino acid sequence presented in Sequence Identification Number: 96 L ; or (iv) V having the amino acid sequence presented in sequence identification number: 97 H and V having the amino acid sequence presented in Sequence Identification Number: 98 L Includes

[0064] In a specific embodiment, the effector-silencing anti-CTLA4 antibody or effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing the amino acid sequence presented in Sequence Identification No.: 73 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 74 L Domain; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 75 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 76 L Domain; (iii) V containing the amino acid sequence presented in sequence identification number: 77 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 78 L Domain; (iv) V containing the amino acid sequence presented in sequence identification number: 79 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 80 L Domain; (v) V containing the amino acid sequence presented in Sequence Identification Number: 81 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 82 L Domain; (vi) V containing the amino acid sequence presented in sequence identification number: 83 HV containing the amino acid sequence presented in Domain and Sequence Identification Number: 84 L Domain; (vii) V containing the amino acid sequence presented in sequence identification number: 85 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 86 L Domain; (viii) V containing the amino acid sequence presented in sequence identification number: 87 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 88 L Domain; (ix) V containing the amino acid sequence presented in sequence identification number: 89 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 90 L Domain; (x) V containing the amino acid sequence presented in Sequence Identification Number: 91 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 92 L Domain; or (xi) V containing the amino acid sequence presented in sequence identification number: 93 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 94 L Includes the domain.

[0065] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment is selected from the group consisting of F(ab), F(ab')2, Fv, and scFv.

[0066] In a further embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises one or more immunoglobulin monovariable domains (ISVDs), and each ISVD is a variable domain (V) of a camel-based heavy chain monovariable antibody. HHV comprising ) but not including, provided that none of the ISVDs include CDR1 containing the amino acid sequence FYGMG (Sequence ID: 69), CDR2 containing the amino acid sequence DIRTSAGRTTYADSVKG (Sequence ID: 70), and CDR3 containing the amino acid EMSGISGWDY (Sequence ID: 71) or EPSGISGWDY (Sequence ID: 72). HH (The above ISVD is as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087588), or V containing 1, 2, or 3 mutations in CDR3 as disclosed in WO2008071447 HH It is excluded that the ISVD comprising the CDR in an embodiment that does not include, and in which one or more ISVDs are fused to or linked to an effector-silencing heterologous HC domain or Fc domain comprising, for example, any one of the effector-silencing antibody HC domain or Fc domain disclosed herein, is not excluded by this proviso.

[0067] The present invention further provides each of the effector-silencing anti-CTLA-4 antibodies disclosed in Table 4-18, provided that the effector-silencing anti-CTLA-4 antibody does not include ipilimumab consisting solely of N297A substitution.

[0068] The present invention further provides a composition 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.

[0069] The present invention further provides an anti-PD-1 antibody comprising the following:

[0070] (a) HC variable domain (V H Heavy chain (HC) and LC variable domain (V) having ) L A light chain (LC) having ), where (i) V H... comprises at least three HC-complementary determining regions (CDRs) of pembrolizumab, and V L ☐ contains at least 3 LC-CDRs of pembrolizumab, or (ii) V H ... comprises at least three HC-CDRs of nivolumab, and V L It contains at least 3 LC-CDRs of nivolumab, or (iii) V H contains at least three HC-CDRs of semiplimab-rwlc, and V L ... comprises at least three LC-CDRs of semiplimab-rwlc, and

[0071] (b) (i) a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an IgG1Fc domain 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 a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iii) an IgG2Fc domain comprising N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S amino acid substitutions or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iv) an IgG1, IgG2, or IgG4Fc domain comprising an IgG4Fc domain comprising an S228P amino acid substitution and N267A, P329G, D265A / N297A amino acid substitution, or a mutated Fc domain comprising 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.

[0072] In another additional embodiment of the above anti-PD-1 antibody, the anti-PD-1 antibody is (i) V having the amino acid sequence presented in sequence identification number: 29 H and V having the amino acid sequence presented in Sequence Identification Number: 30 L, (ii) V having the amino acid sequence presented in sequence identification number: 23 H and V having the amino acid sequence presented in Sequence Identification Number: 24 L , or (iii) V having the amino acid sequence presented in sequence identification number: 99 H and V having the amino acid sequence presented in Sequence Identification Number: 100 L Includes

[0073] In certain embodiments of the anti-PD-1 antibody, the IgG1, IgG2, or IgG4Fc domain as disclosed herein may additionally contain C-terminal lysine or lack C-terminal lysine or C-terminal glycine-lysine dipeptide.

[0074] The present invention relates to an HC variable domain (V H Heavy chain (HC) and LC variable domain (V) having ) L The present invention further provides an anti-PD-1 antibody fragment comprising a light chain (LC) having ), wherein (i) V H ... comprises at least three HC-complementary determining regions (CDRs) of pembrolizumab, and V L ☐ contains at least 3 LC-CDRs of pembrolizumab, or (ii) V H ... comprises at least three HC-CDRs of nivolumab, and V L It contains at least 3 LC-CDRs of nivolumab, or (iii) V H contains at least three HC-CDRs of semiplimab-rwlc, and V L It contains at least three LC-CDRs of semiplimab-rwlc.

[0075] In another additional embodiment of the anti-PD-1 antibody fragment, the anti-PD-1 antibody fragment is (i) V having the amino acid sequence presented in sequence identification number: 29 H and V having the amino acid sequence presented in Sequence Identification Number: 30 L , (ii) V having the amino acid sequence presented in sequence identification number: 23H and V having the amino acid sequence presented in Sequence Identification Number: 24 L , or (iii) V having the amino acid sequence presented in sequence identification number: 99 H and V having the amino acid sequence presented in Sequence Identification Number: 100 L Includes

[0076] In a specific embodiment of the above anti-PD-1 antibody fragment, the anti-PD-1 antibody fragment is selected from the group consisting of F(ab), F(ab')2, Fv, and scFv.

[0077] The present invention further provides each of the anti-PD-1 antibodies disclosed in Tables 19-27.

[0078] The present invention further provides a composition comprising an anti-PD-1 antibody or an anti-PD-1 antibody fragment as disclosed herein and a pharmaceutically acceptable carrier.

[0079] The present invention further provides an anti-PD-L1 antibody comprising the following:

[0080] (a) HC variable domain (V H Heavy chain (HC) and LC variable domain (V) having ) L A light chain (LC) having ), where (i) V H ... comprises at least three HC-complementary determining regions (CDRs) of durvalumab, and V L ☐ contains at least 3 LC-CDRs of durvalumab, or (ii) V H It contains at least three HC-CDRs of avelumab, and V L It contains at least three LC-CDRs of avelumab, or (iii) V H ... comprises at least three HC-CDRs of atezolizumab, and V L ... comprises at least three LC-CDRs of atezolizumab, and

[0081] (b) (i) a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or a mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an IgG1Fc domain 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 a mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (iii) an IgG2Fc domain comprising N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S amino acid substitutions or a mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iv) an IgG1, IgG2, or IgG4Fc domain comprising an IgG4Fc domain comprising an S228P amino acid substitution and N267A, P329G, D265A / N297A amino acid substitution, or a mutated Fc domain comprising 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, provided that V H and V L In the case of having the amino acid sequences of sequence identification number: 107 and sequence identification number: 108, respectively, the heavy chain (HC) constant domain is not an IgG1 isotype having the N297A / D356E / L358M combination of substitutions, or V H and V LIn the case of having the amino acid sequences of sequence identification number: 103 and sequence identification number: 104, respectively, the HC constant domain is not an IgG1 isotype having the combination of substitutions L234F / L235E / P331S / D356E / L358M.

[0082] In another additional embodiment of the above anti-PD-L1 antibody, the anti-PD-L1 antibody is (i) V having the amino acid sequence presented in sequence identification number: 103 H and V having the amino acid sequence presented in Sequence Identification Number: 104 L , (ii) V having the amino acid sequence presented in sequence identification number: 105 H and V having the amino acid sequence presented in Sequence Identification Number: 106 L , or (iii) V having the amino acid sequence presented in sequence identification number: 107 H and V having the amino acid sequence presented in Sequence Identification Number: 108 L Includes

[0083] In certain embodiments of the anti-PD-L1 antibody, the IgG1, IgG2, or IgG4Fc domain as disclosed herein may additionally contain C-terminal lysine or lack C-terminal lysine or C-terminal glycine-lysine dipeptide.

[0084] The present invention relates to an HC variable domain (V H Heavy chain (HC) and LC variable domain (V) having ) L Further providing an anti-PD-L1 antibody fragment comprising a light chain (LC) having ) where (i) V H ... comprises at least three HC-complementary determining regions (CDRs) of durvalumab, and V L ☐ contains at least 3 LC-CDRs of durvalumab, or (ii) V H It contains at least three HC-CDRs of avelumab, and V L It contains at least three LC-CDRs of avelumab, or (iii) V H... comprises at least three HC-CDRs of atezolizumab, and V L It contains at least three LC-CDRs of atezolizumab.

[0085] In another additional embodiment, the anti-PD-L1 antibody fragment is (i) V having the amino acid sequence presented in sequence identification number: 103 H and V having the amino acid sequence presented in Sequence Identification Number: 104 L , (ii) V having the amino acid sequence presented in sequence identification number: 105 H and V having the amino acid sequence presented in Sequence Identification Number: 106 L , or (iii) V having the amino acid sequence presented in sequence identification number: 107 H and V having the amino acid sequence presented in Sequence Identification Number: 108 L Includes

[0086] In a specific embodiment of the above anti-PD-L1 antibody fragment, the anti-PD-1 antibody fragment is selected from the group consisting of F(ab), F(ab')2, Fv, and scFv.

[0087] The present invention further provides each of the anti-PD-L1 antibodies disclosed in Tables 28-36, provided, however, V H and V L In the case of having the amino acid sequences of sequence identification number: 107 and sequence identification number: 108, respectively, the heavy chain (HC) constant domain is not an IgG1 isotype having the N297A / D356E / L358M combination of substitutions, or V H and V L In the case of having the amino acid sequences of sequence identification number: 103 and sequence identification number: 104, respectively, the HC constant domain is not an IgG1 isotype having the combination of substitutions L234F / L235E / P331S / D356E / L358M.

[0088] The present invention further provides a composition comprising the anti-PD-L1 antibody or anti-PD-L1 antibody fragment disclosed herein and a pharmaceutically acceptable carrier.

[0089] The present invention further provides a composition 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.

[0090] The present invention further provides a composition 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.

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

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

[0093] The present invention further provides any one of an anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody or composition as disclosed herein for the treatment of cancer in an individual or for the manufacture of a medicine for the treatment of cancer in an individual.

[0094] The present invention further provides any one of an anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody fragment or composition as disclosed herein for the treatment of cancer in an individual or for the manufacture of a medicine for the treatment of cancer in an individual.

[0095] In certain embodiments, cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, kidney cancer, Hodgkin 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.

[0096] In certain embodiments, cancer is pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, cancer of the uterus or endometrium, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, cancer of the small intestine or appendix, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. Brief explanation of the drawing

[0097] Figures 1a-1e: CTLA-4 blockade-mediated colitis is Fc-dependent. Balb / c mice were treated with antibodies twice weekly for 55 days as indicated. Figure 1a: Profiling of intestinal inflammation gene expression after administration of the Fc-qualified anti-CTLA-4 antibody (α-CTLA4) or the effector-silencing anti-CTLA-4 antibody with D265A substitution (α-CTLA4 (D265S)). After 7 weeks of twice-weekly treatment, proximal small intestines were collected for evaluation of intestinal inflammation markers by reverse transcription-quantitative polymerase chain reaction (PCR). Heatmaps of the fold change in expression of intestinal inflammation genes for the two antibodies compared to isotype control treatment are presented. Expression was analyzed across multiple panels, and cycle threshold data were normalized to ubiquitin within each panel. Normalized data from genes analyzed as part of the multiple panels were averaged before determining the fold change relative to the isotype control. Fig. 1b: Weight loss over the course of the experiment. Fig. 1c: Intestinal permeability was evaluated by measuring FITC-dextran fluorescence in serum on days 49 and 50. Fig. 1d: Histological findings regarding relative intestinal inflammation and the severity of colitis were examined and scored by a certified pathologist. Fig. 1e: Representative micrographs of hematoxylin and eosin (H&E) stained histological sections of the colon starting from day 55. Figs. 2a-2e: Characterization of CTLA-4 ISVD (nAb). Fig. 2a: Comparison of effector-qualified α-CTLA4 and effector-silencing CTLA4 ISVD (CTLA4 nAb); Fig. 2b: Spleen-activated T cells were cultured for 3 days in the presence of CLTA4 nAb or α-CTLA4 as indicated. Proliferation (Fig. 2c), production of IFNγ (Fig. 2d), and IL-2 (Fig. 22e) were measured and plotted as fold change against isotype controls (mouse IgG2a). Data represent 2-3 independent experiments. Figs. 3a-3d: CTLA4 nAbs combined with anti-PD-1 antibodies (α-PD1) possess potent antitumor efficacy. Fig. 3a: CT26 tumor-bearing mice were administered 20 mpk of the indicated antibodies (α-CTLA4, α-PD1, α-CTLA4 (D265A)) and / or 30 mpk of CTLA4 nAb every 4 days for 5 doses when the tumor reached an average size of 100 mm³ (range 78–125 mm³). Data present the average tumor volume over a 32-day period. Results represent two independent studies (n=10 mice per group); Fig. 3b: CD8 T cells / Foxp3+ T cells in the tumor on day 1 after treatment as indicated. reg B. Results represent two independent experiments (n=7 mice per group); Figs. 3c and 3d: Gene expression profiles from whole tumors at day 8 after treatment. Results represent one or two independent experiments (n=5 mice per group). * p<0.05, **p<0.01, *** p<0.001 (independent samples t-test). Error bars ± SEM. Fig. 3e: Individual animal tumor volumes for each treatment group compared to isotype controls. Complete response (CR) up to day 39 is presented for the responsive treatment groups. Data represent two independent experiments using n=10 mice per group. Figures 4a-4d: Anti-CTLA-4 antibody-mediated colitis is Fc-dependent. Balb / c mice were treated twice a week for 55 days with antibodies (α-CTLA4, α-PD1, α-CTLA4 (D265A)) alone or in combination with CTLA4 nAb as indicated. Figure 4a: Body weight loss over the course of the experiment; Figure 4b: Histological evaluation of colitis in the proximal jejunum on day 55; Figure 4c: Micrograph of H&E-stained histological sections of the colon; Figure 4d: Presents a heatmap of the ploid change in expression of intestinal inflammation genes for the presented samples compared to isotype control treatment. Expression was analyzed across multiple panels, and cycle threshold data were normalized to ubiquitin within each panel. Normalized data from the genes analyzed as part of the multiple panels were averaged before determining the ploid change relative to the isotype control. Figs. 5a-5c: Fc effector function anti-CTLA-4 induces cutaneous inflammation but not systemic inflammation. Balb / c mice were treated with α-CTLA4 or α-CTLA4 (D265A) twice a week for 55 days as indicated. Fig. 5a: Microscopic image of H&E-stained histological section of ear skin. Fig. 5b: Ear skin IL-17-producing T cells, Foxp3+ T reg The absolute number of cells and neutrophils was measured by flow cytometry. Fig. 5c: Microscopic images of H&E-stained histological sections of the kidney (top panel), liver (middle panel), and lung (bottom panel). Results represent one of two independent experiments (n=4–8 mice per group). The scale bar indicates 100 μm. Error bars ± SEM. Figs. 6a-6d: Fc-sufficient anti-CTLA-4 antibody against colon Foxp3+ T reg It does not deplete. Fig. 6a: Intracellular CTLA-4 staining of CT26-tumor-bearing mice in the indicated organ. Fig. 6b: Foxp3+ T regMean fluorescence intensity (MFI) of CTLA-4 on cells. **p<0.01, ***p<0.001 (paired-sample t-test). Figs. 6c and 6d: Colon lamina propria and CT26 tumor infiltration Foxp3+ T at 24 hours after treatment as indicated. reg Representative dot plots and statistics. Data represent 2 to 4 independent experiments (n=4–12 mice per group). **p<0.01, ***p<0.001 (independent samples t-test). Error bars ± SEM. Figs. 7a-7d: Fc-function-mediated intestinal response to anti-CTLA-4 in the T of colitis reg - Impaired mediated inhibition. Spleen CD45Rb high Naive T cells were delivered to CB17-SCID recipient mice and treated with α-CTLA4 or CTLA4 nAb as indicated. Fig. 7a: Body weight loss over experimental time. Fig. 7b: Micrograph of H&E-stained histological sections of the colon and Fig. 7c: Pathology scores at day 47 (n=14–18 mice per group). Fig. 7d: Gene expression profile of the whole colon at day 47 after naive T cell delivery (n=6 mice per group). Data represent one of two independent experiments. Ns=Not significant **** p<0.0001 (Independent samples t-test). Error bars ± SEM. Figures 8a-8e: FcγR binding and CTLA-4 blockade activate colonic macrophages. Figures 8a and 8b: CD16 / CD32 surface expression on macrophages isolated from the spleen, colonic lamina propria, and tumors of CT26-retaining mice was evaluated by flow cytometry. Figure 8c: The ratio of macrophages (CD45+CD11b+F4 / 80+) in the spleen, colonic lamina propria, and tumors of CT26-retaining mice was evaluated by flow cytometry. Figure 8d: Il1b, Tnfα, Ifnγ, and Stat1 mRNA expression in the colon of mice treated with α-CTLA4, α-CTLA4 (D265A), or CTLA4 nAb at days 0, 10, and 18 after treatment was evaluated. Data represent two independent experiments (n=8–10 mice per group). ns = not significant, **p<0.01, ***p<0.001 (paired-sample t-test). Error bars ± SEM. Fig. 8e: Proportion of colonic lamina propria IL-17-producing CD4+ T cells (CD45+TCRb+CD4+CD8a-IL-17A+), IFNγ-producing CD8a+ T cells (CD45+TCRb+CD4-CD8a+IFNγ+), and neutrophils (CD45+CD11b+Ly6G high The absolute number of ) was measured by flow cytometry. Results are representative of one of two independent experiments (n=4–8 mice per group). **p<0.01 (independent samples t-test). Scale bars represent 100 μm. Error bars ± SEM. Figures 9a-9b: Antitumor efficacy in a mouse synaptic MB49 bladder tumor model study. Figure 9a: MB49 tumor-bearing mice were administered the indicated antibody doses (30 mg / kg CTLA-4 nAb, 10 mg / kg α-CTLA4, 5 mg / kg α-PD1, or a combination of CTLA4 nAb and α-PD1) every 4 days for 4 doses when the tumor reached an average size of 102 mm³ (range 87-117 mm³). Data present the average tumor volume over a 21-day period. Results represent two independent experiments (n=10 mice per group). Figure 9b: Individual animal tumor volumes for each treatment group are presented. Complete response (CR) up to day 21 is presented for the responsive treatment group. Data present results from experiments using n=10 mice per group. Figs. 10a-10b: Antitumor efficacy in a mouse synaptic MC38 colon tumor model study. Fig. 10a: MC38 tumor-bearing mice were administered the indicated doses of the antibodies (30 mg / kg CTLA-4 nAb, 10 mg / kg α-CTLA4, 5 mg / kg α-PD1, or a combination of CTLA4 nAb and α-PD1) every 4 days for 4 doses when the tumor reached an average size of 220 mm³ (range 179–261 mm³). Data present the average tumor volume over a 23-day period. Results represent two independent experiments (n=10 mice per group). Fig. 10b: Individual animal tumor volumes for each treatment group are presented. Complete response (CR) up to day 23 is presented for the responsive treatment group. Data present results from experiments using n=10 mice per group. Fig. 11: Induction of intestinal inflammation by effector cells. The cartoon illustrating Fc-mediated induction of intestinal inflammation is T reg It can be induced by effector T cells independently of depletion. Specific details for implementing the invention

[0098] definition

[0099] "Adverse Event" or "AE" as used herein refers to any adverse and unintended sign (including abnormal laboratory findings), symptom, or condition transiently associated with the use of a medical treatment or procedure in a human individual, which may or may not be considered associated with the medical treatment or procedure, as presented in the Common Criteria for Adverse Events (CTCAE) Version 5.0, issued by the U.S. Department of Health and Human Services on November 27, 2017. An AE is a term that serves as a unique identifier for a specific event used in medical documentation and scientific analysis. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. The severity of an AE is assigned a grade. Based on these general guidelines, the CTCAE designates grades 1 through 5 for each AE, along with a unique clinical description of the severity: Grade 1, mild, or asymptomatic or mild symptoms, clinical or diagnostic observation only, or no intervention required; Grade 2, moderate, or requiring minimal, local, or non-invasive intervention, or age-appropriate limitation of instrumental activities of daily living (ADL); Grade 3, severe or medically significant but not immediately life-threatening, or requiring hospitalization or extended hospitalization, or incapacity, or limitation of self-care (ADL); Grade 4, life-threatening outcome or requiring urgent intervention; and Grade 5, death related to AE.

[0100] The term “antibody” as used herein refers to a glycoprotein comprising (a) at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide binding, or (b) in the case of camelid antibody species, at least two heavy chains (HC) interconnected by disulfide binding. Each HC consists of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of three domains, C H 1, C H 2 and C H It consists of 3. Generally, the basic antibody structural unit is a tetramer containing two HC / LC pairs, except for camelid antibody species containing only two HCs, in which case the structural unit is a homomer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and one HC chain (about 50-70 kDa).

[0101] In certain naturally occurring antibodies, each light chain is an LC variable region or domain (V L It consists of ) and LC invariant domains. The LC invariant domain is one domain, C L It consists of. Human V H 6 family members: V H 1, V H 2, V H 3, V H 4, V H 5 and V H Includes 6; human V L It has 16 family members: V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, Vλ 6, V λ 7, V λ 8, V λ 9 and V λ Includes 10. Each of these family members can be further divided into specific subtypes. V H and V L The domain can be further subdivided into a hypervariable region called the Complementarity Decision Region (CDR), which is interspersed with a more conserved region called the Framework Region (FR). Each V H and V L It consists of three CDR regions and four FR regions arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0102] The variable regions of the heavy and light chains contain binding domains that include CDRs interacting with antigens. The constant region of the antibody 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 assignment of amino acids to each domain is generally [Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md. ; 5th ed.; 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. It follows the definition of [Chothia, et al., (1989) Nature 342:878-883] or [196:901-917].

[0103] Typically, amino acid numbering in the heavy chain constant domain begins at number 118, following the Eu numbering scheme. The Eu numbering scheme is based on the amino acid sequence of human IgG1 (Eu), which has a constant domain starting at amino acid position 118 of the IgG1 amino acid sequence described in the literature [Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969)], and the literature [Beranger, et al., Ed. accessible at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#r). IgG1, IgG2, IgG3, and IgG4 constant domains are presented in Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG] (Created: 17 / 05 / 2001, Version: 08 / 06 / 2016).

[0104] Generally, the V of the antibody H / V L The pair consists of 6 CDRs, V H 3 CDRs and V on LAlthough it contains three CDRs, modern technology recognizes that in most cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of generating specific antibodies based solely on the heavy chain CDR3 are known in the relevant art (see, 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. Refer to [Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987)] (defining the CDR region of an antibody by its sequence); also refer to [Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987)] (defining the CDR region of an antibody by its structure).

[0105] The following general rules, disclosed in the literature [www.bioinf.org.uk : Prof. Andrew CR Martin's Group] and reproduced in the table below, can be used to identify CDRs in antibody sequences containing amino acids that specifically interact with amino acids containing epitopes in the antigen to which the antibody binds. Although there are rare instances where these generally consistent features do not occur; Cys residues are the most conserved features.

[0106]

[0107]

[0108] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and one HC chain (about 50–70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the HC may define a constant region primarily responsible for the antibody's effector function. Typically, human LCs are classified as kappa and lambda LCs. Additionally, human HCs are typically classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In LC and HC, the variable and constant regions are connected by a “J” region of about 12 or more amino acids, and HC also includes a “D” region of about 10 or more amino acids. Generally, refer to the literature [Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)].

[0109] The heavy chain of the antibody may or may not have a terminal lysine (K) residue, or a terminal glycine and lysine (GK) residue. Therefore, in certain embodiments, the antibody comprises the heavy chain constant region amino acid sequence presented herein which further lacks terminal lysine and terminates with a glycine residue, or additional embodiments in which terminal glycine residue is also lacked. This is because terminal lysine and sometimes glycine and lysine together may be cleaved during antibody expression or, when introduced into the human body, without apparent adverse effects on antibody efficacy, stability, or immunogenicity. In some cases, the nucleic acid molecule encoding the heavy chain may intentionally omit the codon encoding terminal lysine or the codon for terminal lysine and glycine.

[0110] As used herein, “antibody fragment” or “antigen-binding fragment” refers to a fragment of a full-length antibody, that is, an antibody fragment smaller than the full length that possesses the ability to specifically bind to an antigen bound by a full-length antibody, which is entirely lacking the Fc domain or lacking a portion of the Fc domain that confers the binding of the antibody to FcγR. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; scFv molecules; nanobodies; and multispecific antibodies formed from antibody fragments.

[0111] "Chimeric antibody" as used herein 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 derived from different species (U.S. Patent No. 4,816,567; and literature [Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)]), or (ii) the first and second antibodies are derived from different isotypes (e.g., a variable domain from an IgG1 antibody and a constant domain from an IgG4 antibody). In one aspect, the variable domain is obtained from a non-human antibody, e.g., a mouse antibody ("parent antibody"), and the constant domain sequence is obtained from a human antibody. In a further aspect, the variable domain is a humanized variable domain from a mouse antibody and a constant domain from a human antibody.

[0112] As used herein, "combination therapy" refers to the treatment of a human or animal individual comprising the sequential or joint administration of a first therapeutic agent and a second therapeutic agent to the individual. Generally, the first and second therapeutic agents are administered to the individual separately rather than as a mixture; however, there may be embodiments in which the first and second therapeutic agents are mixed prior to administration.

[0113] "Conservative substitution" as used herein means that amino acids can be frequently substituted with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone type, and stiffness, etc.) without altering the biological activity of the protein. Those skilled in the art generally recognize that a single amino acid substitution in the non-essential region of a polypeptide does not substantially alter biological activity (see, for example, the literature [Watson et al. Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.) (1987)]). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to impair biological activity. Exemplary conservative substitutions are presented in Table 2.

[0114]

[0115] "Cytotoxic T lymphocyte-associated antigen-4", "CTLA-4", "CTLA4", "CTLA-4 antigen", and "CD152" (see, e.g., [Murata, Am. J. Pathol. 155:453-460 (1999)]) are used interchangeably and include variants, isoforms, species homologs of human CTLA-4, and analogs having at least one common epitope with CTLA-4 (see, e.g., [Balzano, Int. J. Cancer Suppl. 7:28-32 (1992)]). The complete CTLA-4 nucleic acid sequence can be found in GenBank accession number L15006.

[0116] As used herein, "effector function" refers to biological activity attributed to the Fc region of an antibody and varying depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Antibodies act by numerous mechanisms, most of which bind to different arms of the immune system. Antibodies can simply block molecular interactions or activate the classical complement pathway (known as complement-dependent cytotoxicity or CDC) through the interaction of clustered antibodies with C1q on the C1 complex. Crucially, antibodies also act as a link between antibody-mediated immune responses and cell-mediated immune responses through binding to Fc receptors.

[0117] "Effector-silencing" as used herein refers to an antibody or antibody fragment exhibiting (i) immeasurable binding to one or more Fc receptors (FcRs) as measurable in a Viacore assay, wherein the association constant in the micromolar range indicates immeasurable binding, or (ii) measurable binding to one or more FcRs as measurable in a Viacore assay, which is reduced compared to typical binding for antibodies of the same isotype. In certain embodiments, the antibody may include one or more mutations in the HC constant domain and the Fc domain such that the mutated antibody, in particular, has reduced or immeasurable binding to FcγRIIIa, FcγRIIa, and FcγRI compared to a wild-type antibody of the same isotype as the mutated antibody. In certain embodiments, the affinity or association constant of the effector-silencing antibody for one or more of FcγRIIIa, FcγRIIa, and FcγRI is reduced by at least 1000-fold compared to the affinity of the wild-type isotype; Compared to the affinity of the wild-type isotype, it is reduced by at least 100 to 1000 times; compared to the affinity of the wild-type isotype, it is reduced by at least 50 to 100 times; compared to the affinity of the wild-type isotype, it is reduced by at least 10 to 50 times. In certain embodiments, the effector-silencing antibody has an undetectable or immeasurable binding to one or more of FcγRIIIa, FcγRIIa, and FcγRI compared to binding by the wild-type isotype. Generally, the effector-silencing antibody will lack measurable antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The effector-silencing antibody fragment will lack the Fc domain or the portion of the Fc domain conferring binding to FcR, and will exhibit an undetectable or immeasurable binding to one or more of FcγRIIIa, FcγRIIa, or FcγRI. In the case of effector-silencing antibodies or antibody fragments, binding is measured against human FcR.

[0118] The "Fab fragment" as used herein is a V of one LC and one HC. H and C H Includes 1 and excludes the remainder of the HC invariant domain. C of the Fab molecule H 1 cannot form a disulfide bond with another Fab fragment or HC-containing molecule. "Fab fragment" may be a product of papain cleavage of the antibody.

[0119] The "Fab' fragment" as used herein is C H 1 and C H V up to the area between the 2 domains H A F(ab')2 molecule can be formed by including a fragment of one LC and one HC containing a domain and an HC constant domain, excluding the remainder of the HC constant domain, and forming an interchain disulfide bond between the two HCs of the two Fab' fragments.

[0120] The "F(ab')2 fragment" as used herein comprises two LC and two HC fragments, and each HC fragment is C H 1 and C H V up to the area between the 2 domains H It contains a domain and an HC constant domain, and excludes the remainder of the HC constant domain so that an interchain disulfide bond is formed between the two HCs. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The F(ab')2 fragment [assists] the antibody C H 1 and C H It can be obtained by digesting the antibody using pepsin that cleaves at the region between the two domains.

[0121] "Fc domain" or "Fc" as used herein refers to the C of the antibody. H 2 and C HIt is a crystallizable fragment domain or region obtained from an antibody containing 3 domains. In the antibody, the two Fc domains are connected by two or more disulfide bonds and C H The three domains are held together by hydrophobic interactions. The Fc domain can be obtained by digesting the antibody with the protease papain.

[0122] "Fc receptor" or "FcR" as used herein is a major immunomodulatory receptor that links antibody-mediated (humoral) immune responses to cell effector functions. Receptors for all classes of immunoglobulins, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD), have been identified. There are three classes of receptors for human IgG found in leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (nanomolar range KD), whereas FcγRII and FcγRIII are low to medium-affinity (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 target cells. Upon binding, signaling pathways are triggered that lead to the secretion of various substances mediating the destruction of target cells, such as lytic enzymes, perforins, granzymes, and tumor necrosis factor. The level of ADCC effector function varies for human IgG subtypes. In simple terms, this depends on allotypes and specific FcRs; however, ADCC effector function is high for human IgG1 and IgG3 and low for IgG2 and IgG4.

[0123] The "Fv region" as used herein is V H polypeptide and V LA single V where polypeptides are held together by disulfide bonds H and V L Includes pairs.

[0124] "Humanization" (also referred to as remodeling or CDR-grafting) as used herein is a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from heterologous sources (typically rodents) and improving effector functions (ADCC, complement activation, C1q binding). Although engineered mAbs are manipulated using molecular biology techniques, simple CDR-grafting of the rodent complementation-determining region (CDR) into the human framework often results in a loss of binding affinity and / or specificity of the original mAb. To humanize antibodies, the design of the humanized antibody involves mutations, such as conservative amino acid substitutions of CDR residues, and reverse substitution (reverse mutation) of residues from the rodent mAb to the human framework region. Locations can be identified or confirmed by sequence comparison for structural analysis or by analysis of homology models of the three-dimensional structure of the variable region. The affinity maturation process utilized a phage library to alter amino acids at the most recently selected locations. Similarly, many approaches have been used to select the most appropriate human framework for grafting rodent CDRs. As datasets of known parameters for antibody structures increase, so too do the sophistication and improvement of these techniques. Consensus or germline sequences from a single antibody, or fragments of framework sequences within the variable regions of each light or heavy chain from several different human mAbs, may be used. Another approach to humanization is to modify only the surface residues of the rodent sequence to the most common residues found in human mAbs, which is called "resurfaceization" or "veneering." Often, human or humanized antibodies are substantially non-immunogenic in humans.

[0125] As used herein, “humanized antibody” refers to a form of antibody or antibody fragment containing sequences from both human and non-human (e.g., murine, rat) antibodies. Generally, the humanized antibody will contain at least one, and typically two, variable domains, wherein the hypervariable loop corresponds to that of the non-human immunoglobulin, and all or substantially all of the framework (FR) domains are of the human immunoglobulin sequence. The humanized antibody may optionally contain at least a portion of the human immunoglobulin constant domain (e.g., the Fc domain).

[0126] "Hyperproliferative disease" as used herein refers to a condition in which cell growth is increased above normal levels. For example, hyperproliferative diseases or disorders include malignant diseases (e.g., esophageal cancer, colon cancer, biliary tract cancer) and non-malignant diseases (e.g., atherosclerosis, benign hyperplasia, benign prostatic hyperplasia).

[0127] "Immune-related adverse event" or "irAE" as used herein refers to an AE, which is an autoimmune manifestation resulting from an imbalance of the immune system that may be caused by the use of one or more immune checkpoint inhibitors, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies.

[0128] The term “immune response” as used herein refers to the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by said cells or liver (including antibodies, cytokines, and complement) that result in the selective damage, destruction, or removal from the human body of, for example, invading pathogens, cells or tissues infected with pathogens, cancerous cells, or in the case of autoimmune or pathological inflammation.

[0129] "Immunoglobulin single variable domain" (also referred to as "ISV" or "ISVD") as used herein is generally used without interaction with another variable domain (e.g., V of a conventional 4-chain monoclonal antibody). H and V L V required between domains H / V L Immunoglobulin variable domains (V) capable of forming functional antigen-binding sites (without interaction) H , V HH or V L It is used to refer to a heavy or light chain domain containing a domain. An example of an ISVD is a nanobody (V HH , Humanization VHH and / or Camelization V H , for example, camelized human V H (including), IgNAR, domain, V H It is a domain or V H Antibodies (single-domain) derived from the domain (e.g., dAb™) and V L It is a domain or V L Includes (single-domain) antibodies (e.g., dAb™) derived from a domain. Heavy chain variable domain (e.g., V H or V HH An ISVD based on and / or derived from the domain is generally preferred.

[0130] As used herein, “monoclonal antibody” refers to a substantially homogeneous population of antibodies, that is, the antibody molecules comprising said population have identical amino acid sequences, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically comprise a number of different antibodies having different amino acid sequences in variable domains that are often specific to different epitopes. The modifier “monoclonal” indicates the characteristics of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of antibodies by any particular method. For example, the monoclonal antibody used according to the present invention may be produced by the hybridoma method first described in the literature [Kohler et al., Nature 256: 495 (1975)] or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in, for example, [Clackson et al., Nature 352: 624-628 (1991)] and [Marks et al., J. Mol. Biol. 222: 581-597 (1991)]. Also refer to [Presta J. Allergy Clin. Immunol. 116:731 (2005)].

[0131] "Nanobody" and "Nanobodyes" as used herein are registered trademarks of Ablynx NV.

[0132] As used herein in relation to antibodies or immunoglobulins, “non-human amino acid sequence” refers to an amino acid sequence that is characteristic of the amino acid sequence of a non-human mammal. The term does not include the amino acid sequence of an antibody or immunoglobulin obtained from a complete human antibody library in which the variety of the library is produced in silico (see, for example, U.S. Patent No. 8,877,688 or 8,691,730).

[0133] "PD-1" refers to the programmed death 1 (PD-1) protein, an inhibitory 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. The PD-1 gene codes for 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 suggested to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is known as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al., EMBO J. 11:3887-3895 (1992); Blank, C. et al., Immunol. Immunother. 56(5):739-745 (Epub 2006 Dec. 29)). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, for example, a reduction in tumor-infiltrating lymphocytes, a decrease 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)).Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1 or PD-L2; the effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al., Proc. Nat'l. Acad. Sci. USA 99:12293-12297 (2002); Brown et al., J. Immunol. 170:1257-66 (2003)).

[0134] "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, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found in GenBank accession number U64863.

[0135] "ScFv" or "single-strand variable fragment" as used herein refers to V fused or connected together by a short linker peptide of 10 to about 25 amino acids. H and V L It is a fusion protein containing. The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility, and V H The N-terminus of V L It can be linked to the C-terminus or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the invariant region and the introduction of a linker.

[0136] As used herein, “subtherapeutic dose” means a dose of a therapeutic compound (e.g., antibody) 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 the 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 the antibody less than about 3 mg / kg, i.e., the known monotherapy dose of the anti-CTLA-4 antibody Yervoy for the treatment of unresectable or metastatic melanoma, or a single dose of Yervoy less than about 10 mg / kg, which is the known monotherapy dose for adjuvant melanoma.

[0137] As used herein, “to treat” or “treating” means administering, either internally or externally, a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments thereof of the present invention, to a subject or patient who has one or more disease symptoms or is suspected of having a disease for which the agent has therapeutic or prophylactic activity. Typically, the agent is administered in an amount effective in alleviating one or more disease symptoms in the subject or population being treated by inducing regression of these symptom(s) or inhibiting their progression to any clinically measurable degree. The amount of therapeutic agent effective in alleviating any specific disease symptom may depend on factors such as the patient’s disease status, age and weight, and the ability of the drug to induce a desired response in the subject. Whether disease symptoms have been alleviated may be assessed by any clinical measurement typically used by a physician or other skilled healthcare provider to evaluate the severity or progression of said symptoms. The above terms further include the delay in the onset of symptoms associated with the disorder and / or a reduction in the severity of the symptoms of such disorder. The above terms further include improving uncontrolled or unwanted existing symptoms, preventing additional symptoms, and improving or preventing the underlying causes of such symptoms. Therefore, the above terms indicate that beneficial results have been conferred upon a human or animal subject having a disorder, disease, or symptom, or having the potential to develop such disorder, disease, or symptom.

[0138] As used herein, "therapeutic effective dose" refers to an amount of a specific substance sufficient to achieve a desired effect in the subject to be treated. For example, this may be the amount of a CTLA-4 blocker required to inhibit CTLA-4 activation and induce an anti-tumor response, or the amount required for enhanced anti-PD-1 or PD-L1 responsiveness when co-administered with an anti-PD-1 or anti-PD-L1 blocker.

[0139] The "therapeutic index" (also known as "therapeutic window," "safety window," or "therapeutic ratio") as used herein is a comparison of the amount of therapeutic agent that causes toxicity and the amount of therapeutic agent that causes therapeutic effect.

[0140] As used herein, “treatment” applied to human or veterinary subjects refers to therapeutic treatment involving contact with an antibody or antigen-binding fragment to a human or animal subject requiring treatment with an antibody or antibody fragment.

[0141] "V as used hereto HH "is V H It indicates that the domain is obtained from, derived from, or originates from HC antibodies. Heavy chain antibodies are functional antibodies that have two HCs and no LCs. Heavy chain antibodies exist in and can be obtained from the Camelidae, a member of the biological family Camelidae.

[0142] Introduction

[0143] PD-1 antagonists, such as the commercially available anti-PD-1 antibodies Keytruda and Opdivo, contain human IgG4 backbones with reduced FcγR function. This is because preclinical studies of anti-PD-1 antibodies with FcγR binding function demonstrated poor antitumor efficacy due to the depletion of CD8+ cytotoxic T cells (CTLs), which is essential for tumor immunotherapy (see, e.g., International Patent Application WO2014 / 089113). In contrast, monotherapy using anti-CTLA-4 antibodies involves mutant mouse IgG1 anti-CTLA-4 antibodies lacking measurable FcγR-binding affinity—which requires FcγR function to elicit a strong antitumor response—and mouse IgG1 with high FcγR-binding affinity. 2a- This was demonstrated in preclinical studies comparing anti-CTLA-4 antibodies (e.g., see literature [Selby et al., Cancer Immunol Res. 1:32-42 (2013)]). In anti-CTLA-4 antagonist monotherapy, the requirements for FcγR function are T in the spleen or lymph nodes. reg T regulatory cells in a murine tumor model (T reg It was correlated with the depletion of ) (Simpson et al., J. Exp. Med. 210:1695-710 (2013)).

[0144] The inventors of the present invention hypothesized that by combining an anti-CTLA-4 antibody with an anti-PD-1 antibody, the requirement for FcγR function for the efficacy of the anti-CTLA-4 antibody can be avoided. This hypothesis relates to depleted CD8 + This is supported by new data illustrating the important role of CD28-mediated co-stimulation in the anti-PD-1-mediated activation of cytotoxic T cells. Anti-CTLA-4 and anti-PD-1 antibodies exert antitumor activity through different mechanisms. Importantly, the combined effect of anti-CTLA-4 and anti-PD-1 antibodies is not merely additive, as the combined blockade exerted by the two antibodies results in the activation of many genes, including proliferation-associated and chemokine genes, which are not activated by the antibodies alone (see, for example, Figures 3c and 4d). These data suggest that the mechanism of action for CTLA-4 blockade in monotherapy differs from its mechanism of action when performed in combination with PD-1 blockade.

[0145] The new data is the effector T (T after PD-1 blocking effThis indicates the importance of CD28-mediated co-stimulation in activating cells. As previously hypothesized, PD-1 signaling dephosphorylates CD28 rather than TCRs, and CD28 signaling is required for the enhanced antitumor response observed after PD-1 blockade. Therefore, while monotherapy CTLA-4 blockade can primarily target T cell priming events, combining CTLA-4 blockade with PD-1 blockade is expected to promote the activation of depleted T cells beyond what is anticipated from PD-1 blockade alone. The inventors suggest that this mechanism enables increased CD28-mediated activation independently of Fc-receptor function, involving CTLA-4 antagonists, and T cells that may play a significant role in irAE-mediated toxicity. reg It was assumed that it is enhanced by cell depletion.

[0146] Subsequently, potential warnings regarding anti-CTLA-4 antibodies (Fc-functional antibodies) binding to FcR are T regIt is suggested that depletion or cellular bridging between T cells and myeloid cells can induce unwanted immune-related inflammation. The inventors hypothesized that Fc function may contribute to the observed irAEs associated with CTLA-4 blockade cancer immunotherapy. One criticism used to counter the potential role of Fc function in the induction of irAEs is that both ipilimumab (for the human IgG1 backbone) and tremelimumab (for the human IgG2 backbone) treatments are associated with intestinal inflammation. Although the human IgG2 Fc domain has a significantly lower affinity for human FcγR compared to human IgG1, direct comparisons of antibodies with human IgG2 and IgG1 backbones have suggested that both induce 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 of a chimeric anti-mouse CTLA-4 antibody having a human IgG1 isotype backbone or a human IgG2 isotype backbone reg Depletion and antitumor activity were equivalent in human FcγR-depleted mice (Vargas et al., ibid.).

[0147] A major obstacle to evaluating the potential role of Fc function in inducing intestinal inflammation in synaptic tumor models was the lack of measurable inflammation and colitis using mouse anti-CTLA-4 surrogate antibodies. To circumvent this obstacle, the inventors utilized a PCR-based panel previously developed by [Cayatte et al., Clin. Transl. Gastroenterol. 3: e10 (2012)] to measure the upregulation of intestinal inflammation genes associated with inflammatory bowel disease (IBD) in mouse IBD models. As presented in the examples herein, using this PCR-based panel, the inventors were able to detect increased expression of biomarker genes indicating intestinal inflammation in mice treated with an Fc-functional anti-mouse CTLA-4 antibody (α-CTLA4) even in the absence of histological evidence of apparent colitis or tissue damage (see Figures 1a, 3c, and 4d). These observations of subclinical stimulation of the intestinal inflammation gene expression pathway encouraged the inventors to extend the treatment schedule to determine whether the underlying inflammation would progress to the development of clinical colitis. These irAE colitis mouse models enabled the inventors to conduct empirical experiments to evaluate the requirements for Fc function for the induction of intestinal inflammation in the absence or context of the accompanying antitumor response in synonymous tumor models used in immuno-oncology preclinical development.

[0148] The results described in the examples clearly indicate that neither the effector-silenced anti-CTLA-4 antibody nor the effector-silenced anti-CTLA-4 antibody fragment induces measurable antitumor activity in a monotherapy setting. However, administering the effector-silenced antibody or effector-silenced antibody fragment in combination with an anti-PD1 antibody results in antitumor activity comparable to that induced by the effector-functional anti-CTLA-4 antibody alone or in combination with an anti-PD-1 antibody (see Fig. 3a), and there were no intestinal or cutaneous irAEs (Figs. 4b and 5a) or weight loss (Fig. 4a) observed for the effector-functional anti-CTLA-4 antibody alone or in combination with an anti-PD-1 antibody. These results and the potential T associated with Fc-mediated antitumor activity and intestinal inflammation reg - In light of the inventors' discovery of an independent mechanism, the present invention enables a CTLA-4 / PD-1 blockade combination anticancer immunotherapy with an improved therapeutic index and broader utility.

[0149] Combination therapy

[0150] The present invention provides an anticancer combination therapy comprising administering to an individual requiring cancer therapy (i) a PD-1 blocker selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an effector-silenced anti-PD-1 antibody, an effector-silenced anti-PD-L1 antibody, an effector-silenced anti-PD-1 antibody fragment, and an effector-silenced anti-PD-L1 antibody fragment; and (ii) an effector-silenced CTLA-4 blocker selected from the group consisting of an effector-silenced anti-CTLA-4 antibody and an effector-silenced anti-CTLA-4 antibody fragment)

[0151] Effector-silencing CTLA-4 blockers may be administered in combination therapy with PD-1 blockers at a dose greater than the therapeutic dose of ipilimumab 1 mg / kg approved by the U.S. FDA for ipilimumab / nivolumab combination therapy targeting advanced renal cell carcinoma or metastatic colorectal cancer with high microsatellite instability or mismatch repair deficiency, and at a lower risk of inducing cutaneous or intestinal irAEs greater than grade 1 or 2 according to the criteria set forth in the Common Criteria for Adverse Events (CTCAE) version 5.0 during the duration of the combination therapy or for at least part of the period the subject is receiving the combination therapy than observed for ipilimumab / nivolumab combination therapy. In certain embodiments, the dose does not induce irAEs greater than grade 1 during the duration of the combination therapy or for at least part of the period the subject is receiving the combination therapy.

[0152] Therefore, in certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose greater than 1 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose of at least 3 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose of at least 10 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose of at least 15 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose of at least 20 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a dose of 3 mg / kg to 20 mg / kg. In certain embodiments, an effector-silent CTLA-4 blocker may be administered to an individual at a fixed dose that does not depend on the individual's body weight, for example, at a dose greater than 100 mg.

[0153] In a specific embodiment of the combination therapy, the effector-silencing CTLA-4 blocker is either (b) an effector-silencing anti-CTLA-4 antibody or (b) an effector-silencing anti-CTLA-4 antibody fragment. Since effector functional activity is not required for the anti-CTLA-4 antibody, the anti-CTLA-4 antibody has an HC domain engineered to be "effector-silencing" (i.e., its Fc domain is modified to have reduced or immeasurable FcR binding compared to the Fc domain of the wild-type antibody of the same isotype as the effector-silencing antibody as determined by Viacor assay (e.g., the Fc domain of a non-mutated IgG1, IgG2, IgG3, or IgG4 Fc domain)). The effector-silencing anti-CTLA-4 antibody fragment lacks the Fc domain or a region of the Fc domain that binds to one or more FcRs.

[0154] In a specific embodiment, the combination therapy of the present invention may be administered to an individual before or after surgery to remove a tumor, and may be used before, during, or after radiation therapy.

[0155] In a specific embodiment, the combination therapy of the present invention is administered to an individual who has never been previously treated with a biotherapeutic agent or a chemotherapeutic agent, i.e., an individual with no prior treatment experience. In another embodiment, the combination therapy is administered to an individual who has not achieved a sustained response after prior therapy using a biotherapeutic agent or a chemotherapeutic agent, i.e., an individual with prior treatment experience.

[0156] In certain embodiments, the combination therapy of the present invention is used to treat tumors large enough to be detected by palpation or by imaging techniques widely known in the art, such as MRI, ultrasound, or CAT scans. In some embodiments, the combination therapy of the present invention is used to treat advanced-stage tumors having dimensions of at least about 200 mm³, 300 mm³, 400 mm³, 500 mm³, 750 mm³, or up to 1000 mm³.

[0157] In certain embodiments, the combination therapy of the present invention is administered to an individual having cancer that tests 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 an immunohistochemistry (IHC) assay on a fixed formalin paraffin embedding (FFPE) or frozen tissue section of a tumor sample removed from the individual. The individual's physician may order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the individual prior to the initiation of treatment using the combination therapy of the present invention, but it is considered that the physician may order a first or subsequent diagnostic test at any time after the initiation of treatment, for example, after the completion of a treatment cycle.

[0158] The combination therapy may include any one of the exemplary anti-PD-1 antibody or anti-PD-1 antibody fragment disclosed herein in combination with any one of the exemplary anti-PD-L1 antibody or anti-PD-L1 antibody fragment disclosed herein.

[0159] (a) Effector-silencing antibody

[0160] The effector-silencing antibody of the present invention comprises an HC constant domain or its Fc domain modified such that the antibody exhibits immeasurable binding to one or more FcRs or reduced binding to one or more FcRs compared to an unmodified antibody of the same IgG isotype. In further embodiments, the effector-silencing antibody may exhibit immeasurable binding to FcγRIIIa, FcγRIIa, and FcγRI, respectively, or reduced binding to FcγRIIIa, FcγRIIa, and FcγRI, respectively, compared to an unmodified antibody of the same IgG isotype. In certain embodiments, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

[0161] In a specific embodiment, the effector-silencing antibody comprises an Fc domain of the IgG1, IgG2, IgG3, or IgG4 isotype modified to lack N-glycosylation of the asparagine (Asn) residue at position 297 (Eu numbering system) of the HC constant domain. The consensus sequence for N-glycosylation is Asn-Xaa-Ser / Thr (where Xaa at position 298 is any amino acid excluding Pro); and for all four isotypes, the N-glycosylation consensus sequence is Asn-Ser-Thr. The modification can be achieved by replacing the codon encoding Asn at position 297 of the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, e.g., Ala, Asp, Gln, Gly, or Glu (e.g., N297A, N297Q, N297G, N297E, or N297D). Alternatively, the codon for Ser at position 298 may be replaced with a codon for Pro, or the codon for Thr at position 299 may be replaced with any codon other than the codon for Ser. In a further alternative, each amino acid containing the N-glycosylation consensus sequence is replaced with another amino acid. These modified IgG molecules have an unmeasurable effector function. In certain embodiments, these mutated HC molecules may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, said substitutions may be conserved or non-conserved mutations. In further embodiments, these IgG modified to lack N-glycosylation at position 297 may further include one or more additional mutations disclosed herein to eliminate a measurable effector function.

[0162] An exemplary IgG1HC constant domain mutated at position 297 to nullify the N-glycosylation of the HC constant domain is presented at sequence identification number: 44, an exemplary IgG2HC constant domain mutated at position 297 to nullify the N-glycosylation of the HC constant domain is presented at sequence identification number: 50, and an exemplary IgG4HC constant domain mutated at position 297 to nullify the N-glycosylation of the HC constant domain is presented at sequence identification number: 56. In certain embodiments, 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, said substitutions may be conserved or non-conserved mutations.

[0163] In a specific embodiment, the Fc domain of the HC constant domain of IgG1, IgG2, IgG3, or IgG4 containing an effector-silencing antibody is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified by Eu numbering), wherein the HC constant domain is effector-silencing. In a specific embodiment, the modified IgG1 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.

[0164] In certain embodiments, the HC constant domain comprises L234A, L235A, and D265S substitutions (wherein positions are identified by Eu numbering). In certain embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one additional amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein positions are identified by Eu numbering). These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; and the literature [Sondermann et al. Nature 406, 267-273 (20 Jul. 2000)].

[0165] In the specific embodiment 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 Eu numbering. In the specific 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.

[0166] In a specific embodiment, the effector-silencing antibody comprises an IgG1 isotype, wherein the Fc domain of the HC constant domain is modified to be effector-silencing by substituting amino acids at positions 233 through 236 of IgG1 with the corresponding amino acids of human IgG2HC and amino acids at positions 327, 330, and 331 with the corresponding amino acids of human IgG4HC, wherein the positions are identified by 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 specific 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 the substitutions may be conserved or non-conserved mutations.

[0167] In certain embodiments, the effector-silencing antibody is fused to or connected to a hybrid human immunoglobulin HC constant domain comprising a hinge region, a CH2 domain, and a CH3 domain in the direction from the N-terminus to the C-terminus. H It comprises a domain, wherein the hinge region comprises at least a partial amino acid sequence of the human IgD hinge region or the human IgG1 hinge region; the CH2 domain is that of the human IgG4CH2 domain, and a portion thereof is replaced at its N-terminal region by 4–37 amino acid residues of the N-terminal region of the human IgG2CH2 or human IgD CH2 domain. Such a hybrid human HC constant domain is disclosed in U.S. Patent No. 7,867,491, the full text of which is incorporated herein by reference.

[0168] In certain embodiments, the effector-silencing antibody comprises an IgG4HC constant domain in which the serine at position 228 according to the Eu system is substituted with proline (see, e.g., SEQ ID No. 52). This modification prevents the formation of potential interchain disulfide bonds between cysteines at positions Cys226 and Cys229 in the EU system and may interfere with the formation of proper intrachain disulfide bonds. See [Angal et al. Mol. Imunol. 30:105 (1993)]; also see [(Schuurman et al., Mol. Immunol. 38: 1-8, (2001); SEQ ID Nos. 14 and 41)]. In further embodiments, the IgG4 constant domain comprises P239G, D265A, or D265A / N297G amino acid substitutions in addition to the S228P substitution, where the positions are identified according to Eu numbering. In the specific embodiment above, the IgG4HC constant domain is a human HC constant domain. In the specific 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.

[0169] An exemplary IgG1HC constant domain comprises an HC constant domain comprising an amino acid sequence selected from the group consisting of amino acid sequences presented in sequence identification numbers: 38, 39, 40, 41, 42, 43, and 44. An exemplary IgG2HC constant domain has an amino acid sequence selected from the group consisting of amino acid sequences presented in sequence identification numbers: 46, 47, 48, and 49. An exemplary IgG4HC constant domain has an amino acid sequence selected from the group consisting of amino acid sequences presented in sequence identification numbers: 53, 54, 55, and 56.

[0170] More specific examples of effector-silencing antibodies are described below, including specific exemplary effector-silencing anti-CTLA-4 antibodies, anti-PD-1 antibodies, and combinations with anti-PD-1 antibodies.

[0171] (b) Exemplary effector-silencing anti-CTLA-4 antibody

[0172] Exemplary effector-silencing anti-CTLA-4 antibodies and compositions comprising these antibodies that may be used in the combination therapy of the present invention comprise any effector-silencing anti-CTLA-4 antibody that binds to CTLA-4 and inhibits CTLA-4 from binding to B7. Specific effector-silencing anti-CTLA-4 antibodies comprise the following effector-silencing anti-CTLA-4 antibodies and a composition comprising any one of these antibodies and a pharmaceutically acceptable carrier.

[0173] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) three HC-CDRs of ipilimumab fused to or linked to an HC constant domain that exhibit immeasurable or reduced binding to FcγRIIIA, FcγRIIA, and FcγRI compared to a polypeptide containing a wild-type IgG constant domain region as determined by the Viacor assay. H and (ii) V comprising three LC-CDRs of ipilimumab fused to or linked to an LC kappa or lambda constant domain L Includes. Three HC-CDRs each include sequence identification number: 4, sequence identification number: 5, and sequence identification number: 6, and three LC-CDRs each include sequence identification number: 1, sequence identification number: 2, and sequence identification number: 3.

[0174] In a further embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) three HC-CDRs of tremelimumab fused to or linked to an HC constant domain that exhibit immeasurable or reduced binding to FcγRIIIA, FcγRIIA, and FcγRI compared to a polypeptide containing a wild-type IgG constant domain region as determined by the Viacor assay. H and (ii) V comprising three LC-CDRs of tremelimumab fused to or linked to an LC kappa or lambda constant domain L Includes. Three HC-CDRs each include sequence identification number: 12, sequence identification number: 13, and sequence identification number: 14, and three LC-CDRs each include sequence identification number: 9, sequence identification number: 10, and sequence identification number: 11.

[0175] In additional embodiments, the effector-silencing anti-CTLA-4 antibody is (i) V of ipilimumab H and V L Domain, (ii) V of tremelimumab H and V LDomain, (iii) V of REGN4659 H and V L Domain, (iv) V of AGEN1884w H and V L The domain, or (v) V of the anti-CTLA-4 antibody clone 2C8 disclosed in international patent application WO2017194265 H and V L Includes the domain. Ipilimumab V H The domain includes the amino acid sequence presented in sequence identification number: 7, and V L The domain contains the amino acid sequence presented in Sequence Identification Number: 8. Tremelimumab V H The domain includes the amino acid sequence presented in sequence identification number: 15, and V L The domain contains the amino acid sequence presented in sequence identification number: 16. REGN4659 V H The domain includes the amino acid sequence presented in sequence identification number: 95, and V L The domain contains the amino acid sequence presented in sequence identification number: 96. AGEN1884w V H The domain includes the amino acid sequence presented in sequence identification number: 97, and V L The domain comprises the amino acid sequence presented in sequence identification number: 98. In a specific 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 conservative or non-conservative mutations.

[0176] In a further embodiment, the effector-silencing anti-CTLA-4 antibody is V of 8D2 / 8D2 (RE). H and V LDomain (see U.S. Published Patent Application No. 20170216433 and International Application WO2018183408), comprising 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15 or 8D2H2L17, wherein V H The domain is fused to or linked to an HC constant domain exhibiting immeasurable or reduced binding to FcγRIIIA, FcγRIIA, and FcγRI compared to a polypeptide containing a wild-type IgG constant domain region as determined by the Viacore assay, and V L The domain is fused to or connected to an LC kappa or lambda invariant domain.

[0177] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody comprises a variant of 8D2 / 8D2 (RE), 8D2H1L1, 8D2H2L2, 8D2H2L15, or 8D2H2L17, wherein V of the variant H In the amino acid sequence, methionine at position 18 is substituted with isoleucine. Therefore, the effector-silencing anti-CTLA-4 antibody against 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 It may include.

[0178] In a further embodiment, the effector-silencing anti-CTLA4 antibody comprises (i) V comprising the amino acid sequence presented in sequence identification number: 73 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 74 L Domain; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 75 HV containing the amino acid sequence presented in Domain and Sequence Identification Number: 76 L Domain; (iii) V containing the amino acid sequence presented in sequence identification number: 77 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 78 L Domain; (iv) V containing the amino acid sequence presented in sequence identification number: 79 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 80 L Domain; (v) V containing the amino acid sequence presented in Sequence Identification Number: 81 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 82 L Domain; (vi) V containing the amino acid sequence presented in sequence identification number: 83 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 84 L Domain; (vii) V containing the amino acid sequence presented in sequence identification number: 85 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 86 L Domain; (viii) V containing the amino acid sequence presented in sequence identification number: 87 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 88 L Domain; (ix) V containing the amino acid sequence presented in sequence identification number: 89 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 90 L Domain; (x) V containing the amino acid sequence presented in Sequence Identification Number: 91 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 92 L Domain; or (xi) V containing the amino acid sequence presented in sequence identification number: 93 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 94 LIt has a domain. In a specific 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 conservative or non-conservative mutations.

[0179] In a further embodiment of the effector-silencing anti-CTLA-4 antibody, V H The domain is fused to or linked to an IgG4HC constant domain or an IgG1, IgG2, or IgG4HC constant domain modified to contain one or more mutations to make the generated anti-CTLA4 antibody effector-silencing.

[0180] In one embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an amino acid substitution mutation selected from the group consisting of 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 a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and an amino acid substitution mutation selected from the group consisting of 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 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, It comprises an IgG1Fc domain having a mutated Fc domain that additionally includes insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0181] In another embodiment, the effector-silencing anti-CTLA-4 antibody comprises (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or an additional amino acid substitution, insertion, and / or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; (ii) an amino acid substitution mutation selected from the group consisting of N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an amino acid substitution mutation selected from the group consisting of a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, and N297A / D265S, D265A, P329G / D265A / N297G, or V234A / G237A / P238S / H268A / V309L / A330S / P331S, or an IgG2Fc domain having a mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions, insertions and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0182] In a further embodiment, the effector-silencing anti-CTLA-4 antibody has an S228P amino acid substitution and comprises: (i) a mutated Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions; or (iii) an IgG4Fc domain further comprising a mutant Fc domain further comprising a mutation of the N-glycosylation site Asn-Xaa-Ser / Thr starting at amino acid position 297 that nullifies N-glycosylation at the N-glycosylation site, an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A / N297A, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

[0183] Table 4-18 provides specific exemplary anti-CTLA-4 antibodies that may be used in combination with anti-PD-1 or anti-PD-L1 antibodies in a regimen for treating individuals with cancer. The invention also provides the antibodies and compositions presented in the table, except ipilimumab consisting solely of an N297A substitution, each composition comprising the antibodies presented in the table and pharmaceutically acceptable carriers, except for compositions comprising ipilimumab consisting solely of an N297A substitution. All HC amino acid substitution positions in Table 4-18 are in accordance with the Eu numbering scheme.

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] (c) Exemplary effector-silencing anti-CTLA-4 antibody fragment

[0194] Exemplary effector-silencing anti-CTLA-4 antibody fragments and compositions comprising the same that may be used in the combination therapy of the present invention comprise any antibody fragment that binds to CTLA-4 and inhibits CTLA-4 from binding to B7. Specific examples of these 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.

[0195] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing three HC-CDRs of ipilimumab H and (ii) V containing three LC-CDRs of ipilimumab L Fv, scFv, F(ab), or F(ab')2 containing. Three HC-CDRs each contain sequence identification number: 1, sequence identification number: 2, and sequence identification number: 3, and three LC-CDRs each contain sequence identification number: 4, sequence identification number: 5, and sequence identification number: 7.

[0196] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing three HC-CDRs of tremelimumab H and (ii) V containing three LC-CDRs of tremelimumab L Includes. Three HC-CDRs each include sequence identification number: 9, sequence identification number: 10, and sequence identification number: 11, and three LC-CDRs each include sequence identification number: 12, sequence identification number: 13, and sequence identification number: 14.

[0197] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment is (i) V of ipilimumab H and V L Domain, (ii) V of tremelimumab H and V L Domain, (iii) V of REGN4659 H and V L Domain, (iv) V of AGEN1884w H and V L The domain, or (v) V of the anti-CTLA-4 antibody clone 2C8 disclosed in international patent application WO2017194265 H and V L Includes the domain. Ipilimumab V H The domain includes the amino acid sequence presented in sequence identification number: 7, and V L The domain contains the amino acid sequence presented in Sequence Identification Number: 8. Tremelimumab V H The domain includes the amino acid sequence presented in sequence identification number: 15, and V L The domain contains the amino acid sequence presented in sequence identification number: 16. REGN4659 V H The domain includes the amino acid sequence presented in sequence identification number: 95, and V L The domain contains the amino acid sequence presented in sequence identification number: 96. AGEN1884w V H The domain includes the amino acid sequence presented in sequence identification number: 97, and V LThe domain contains the amino acid sequence presented in sequence identification number: 98.

[0198] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment is V of ipilimumab H and V L , V of tremelimumab H and V L , REGN4659's V H and V L , V of AGEN1884w H and V L , V of 8D2 / 8D2 (RE) H and V L , V of 8D2H1L1 H and V L , V of 8D2H2L2 H and V L , V of 8D3H3L3 H and V L , V of 8D2H2L15 H and V L , or V of 8D2H2L17 H and V L Includes

[0199] In a specific embodiment, the anti-CTLA-4 antibody or anti-CTLA-4 antibody fragment is 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 Includes

[0200] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises (i) V containing the amino acid sequence presented in Sequence Identification No.: 73 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 74 LDomain; (ii) V containing the amino acid sequence presented in Sequence Identification Number: 75 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 76 L Domain; (iii) V containing the amino acid sequence presented in sequence identification number: 77 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 78 L Domain; (iv) V containing the amino acid sequence presented in sequence identification number: 79 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 80 L Domain; (v) V containing the amino acid sequence presented in Sequence Identification Number: 81 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 82 L Domain; (vi) V containing the amino acid sequence presented in sequence identification number: 83 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 84 L Domain; (vii) V containing the amino acid sequence presented in sequence identification number: 85 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 86 L Domain; (viii) V containing the amino acid sequence presented in sequence identification number: 87 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 88 L Domain; (ix) V containing the amino acid sequence presented in sequence identification number: 89 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 90 L Domain; (x) V containing the amino acid sequence presented in Sequence Identification Number: 91 H V containing the amino acid sequence presented in Domain and Sequence Identification Number: 92 L Domain; or (xi) V containing the amino acid sequence presented in sequence identification number: 93 HV containing the amino acid sequence presented in Domain and Sequence Identification Number: 94 L Includes the domain.

[0201] In a specific embodiment, the effector-silencing anti-CTLA-4 antibody fragment comprises one or more immunoglobulin monovariable domains (ISVDs), and each ISVD is a variable domain (V) of a camel-based heavy chain monoantibody. HH Includes ) but; However, the ISVD does not include 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) (the said ISVD is as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087588), the ISVD variant contains one, two, or three mutations in CDR3 as disclosed in WO2008071447, and said CDR comprises said CDR in an embodiment in which one or more ISVDs are fused or linked to an effector-silencing antibody constant domain or an Fc domain, e.g., any of the effector-silencing antibody constant domains or Fc domains disclosed herein. The fact that ISVD is not excluded by this proviso is excluded.

[0202] (d) Exemplary anti-PD-1 antibody

[0203] An exemplary anti-PD-1 antibody that can be used in the combination therapy of the present invention comprises any antibody that binds to PD-1 and inhibits PD-1 from binding to PD-L1. In a further embodiment, an exemplary anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and cemiflimab-rwlc. An exemplary antibody comprises the following anti-PD-1 antibody and a composition comprising an anti-PD-1 antibody and a pharmaceutically acceptable salt.

[0204] Pembrolizumab, also known as Keytruda, rembrolizumab, MK-3475, or SCH-900475, is a humanized anti-PD-1 antibody described in U.S. Patent No. 8,354,509 and WO2009 / 114335 and disclosed, for example, in the literature [Hamid, et al., New England J. Med. 369 (2): 134-144 (2013)]. The heavy and light chains for pembrolizumab are represented by the amino acid sequences presented in sequence identification numbers: 27 and 28, respectively.

[0205] 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 for nivolumab are represented by the amino acid sequences presented in Sequence Identification Nos. 25 and 26, respectively.

[0206] Semiplimab-rwlc, also known as semiplimab, 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 for semiplimab-rwlc are represented by the amino acid sequences presented in SEQ ID NOs: 101 and 102, respectively.

[0207] In a specific embodiment, the anti-PD-1 antibody comprises (i) three HC-CDRs of pembrolizumab fused to or linked to an effector-silencing HC constant domain V H and (ii) V comprising three LC-CDRs of pembrolizumab fused to or linked to an LC kappa or lambda constant domain L Includes. Three HC-CDRs each include sequence identification number: 31, sequence identification number: 32 and sequence identification number: 33, and three LC-CDRs each include sequence identification number: 34, sequence identification number: 35 and sequence identification number: 36.

[0208] In a specific embodiment, the anti-PD-1 antibody comprises (i) three HC-CDRs of nivolumab fused to or linked to an effector-silencing HC constant domain V H and (ii) V comprising three LC-CDRs of nivolumab fused to or linked to an LC kappa or lambda constant domain L Includes. Three HC-CDRs each include sequence identification number: 17, sequence identification number: 18, and sequence identification number: 19, and three LC-CDRs each include sequence identification number: 20, sequence identification number: 21, and sequence identification number: 2.

[0209] In a specific embodiment, the anti-PD-1 antibody comprises (i) three HC-CDRs of semiplimab-rwlc fused to or linked to an effector-silencing HC constant domain V H and (ii) V comprising three LC-CDRs of nivolumab fused to or linked to an LC kappa or lambda constant domain L Includes

[0210] In a specific embodiment, the anti-PD-1 antibody is (i) V of pembrolizumab H and V L Domain (here V H The domain is fused to or connected to the effector-silence HC invariant domain, and V L (ii) The domain is fused to or linked to the LC kappa or lambda invariant domain); (ii) V of nivolumab H and V L Domain (here V H The domain is fused to or connected to the effector-silence HC invariant domain, and V L The domain is fused to or linked to the LC kappa or lambda invariant domain); or (iii) V of semiplimab-rwlc H and V L Domain (here V H The domain is fused to or connected to the effector-silence HC invariant domain, and V LThe domain includes (fused to or linked to the LC kappa or lambda invariant domain). Pembrolizumab V H The domain includes the amino acid sequence presented in sequence identification number: 29, and V L The domain contains the amino acid sequence presented in sequence identification number: 30. Nivolumab V H The domain includes the amino acid sequence presented in sequence identification number: 23, and V L The domain contains the amino acid sequence presented in sequence identification number: 24. Semiplimab-rwlc V H The domain includes the amino acid sequence presented in sequence identification number: 99, and V L The domain comprises the amino acid sequence presented in sequence identification number: 100. In a specific embodiment, V H and V L The domain may additionally 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 conservative or non-conservative mutations.

[0211] In a specific embodiment, anti-PD-1 antibody V H The domain is currently a specific V H It may be fused to or linked to an IgG1, IgG2, IgG3, or IgG4HC constant domain that is not linked to, or linked to an IgG1, IgG2, IgG3, or IgG4HC constant domain modified to include one or more mutations in the Fc domain that silence the generated anti-PD-1 antibody.

[0212] In certain embodiments, the HC constant domain is of the IgG1, IgG2, IgG3, or IgG4 isotype, which is modified to lack N-glycosylation of the asparagine (Asn) residue at position 297 of the HC constant domain by replacing the codon for Asn at position 297 of the nucleic acid molecule encoding the HC constant domain with a codon for another amino acid, e.g., Gln. In further embodiments, this IgG modified to lack N-glycosylation at position 297 further comprises one or more additional mutations disclosed herein to remove detectable effector function. In certain embodiments, the HC constant domain is a human HC constant domain. In certain embodiments, the molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, said substitutions may be conserved or non-conserved mutations.

[0213] In a specific embodiment, the present invention provides an anti-PD-1 antibody comprising an IgG4HC constant domain modified to have an S228P substitution and, in addition to the S228P substitution, further include a P239G, D265A, or D265A / N297G amino acid substitution, wherein the positions are identified according to Eu numbering. In the specific embodiment above, the IgG4HC constant domain is a human HC constant domain. In a specific embodiment, the molecule 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.

[0214] In another embodiment, the anti-PD-1 antibody may comprise a human IgG1 isotype, wherein the Fc domain of the HC constant domain is modified to be effector-silencing by substituting the amino acids at positions 233 through 236 of IgG1 with the corresponding amino acids of human IgG2HC and the amino acids at positions 327, 330 and 331 with the corresponding amino acids of human IgG4HC, wherein the positions are identified according to Eu numbering. In a specific 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.

[0215] In another embodiment, the Fc domain of the IgG1, IgG2, IgG3, or IgG4HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, wherein the polypeptide exhibits immeasurable or reduced binding to FcγRIIIA, FcγRIIA, and FcγRI compared to a polypeptide containing the wild-type IgG constant domain region as determined by a ViaCore assay. These and other substitutions are WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; and the literature [Sondermann et al. This is disclosed in Nature 406, 267-273 (20 Jul. 2000). In certain 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.

[0216] Tables 19-27 provide specific exemplary anti-PD-1 antibodies that may be used in combination with anti-CTLA-4 antibodies as disclosed herein in a regimen for treating individuals with cancer. The invention also provides the antibodies and compositions presented in the tables, each composition comprising the antibodies presented in the tables and a pharmaceutically acceptable carrier. All HC amino acid substitution positions in Tables 19-27 are in accordance with the Eu numbering scheme.

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] (e) Exemplary anti-PD-1 antibody fragment

[0223] An exemplary anti-PD-1 antibody fragment that can be used in the combination therapy of the present invention comprises any anti-PD-1 antibody fragment that binds to PD-1 and inhibits PD-1 from binding to PD-L1, and further comprises a composition comprising the following anti-PD-1 antibody fragment that binds to PD-1 and the following anti-PD-1 antibody fragment and a pharmaceutically acceptable carrier.

[0224] In a specific embodiment, the antibody fragment is pembrolizumab V having the amino acid sequence presented in SEQ ID No. 29. H and pembrolizumab V having the amino acid sequence presented in Sequence Identification Number: 30 L It is an Fv or scFv containing.

[0225] In a specific embodiment, the anti-PD-1 antibody fragment is pembrolizumab V having the amino acid sequence presented in SEQ ID No. 29. H and pembrolizumab V having the amino acid sequence presented in Sequence Identification Number: 30 H It is F(ab) containing .

[0226] In a specific embodiment, the anti-PD-1 antibody fragment is pembrolizumab V having the amino acid sequence presented in SEQ ID No. 29. H and pembrolizumab V having the amino acid sequence presented in Sequence Identification Number: 30 H It is F(ab')2 containing .

[0227] In a specific embodiment, the anti-PD-1 antibody fragment is nivolumab V having the amino acid sequence presented in SEQ ID No. 23. H and nivolumab V having the amino acid sequence presented in sequence identification number: 24 H It is an Fv or scFv containing .

[0228] In a specific embodiment, the anti-PD-1 antibody fragment is nivolumab V having the amino acid sequence presented in SEQ ID No. 23. H and nivolumab V having the amino acid sequence presented in sequence identification number: 24 H It is F(ab) containing .

[0229] In a specific embodiment, the anti-PD-1 antibody fragment is nivolumab V having the amino acid sequence presented in SEQ ID No. 23. H and nivolumab V having the amino acid sequence presented in sequence identification number: 24 H It is F(ab')2 containing .

[0230] In a specific embodiment, the anti-PD-1 antibody fragment comprises one or more immunoglobulin monovariable domains (ISVDs), and each ISVD is a variable domain (V) of a camel-based heavy chain monoantibody. HHIncludes ) but; However, the ISVD does not include CDR1 containing the amino sequence THAMG (SEQ ID No.: 73), CDR2 containing the amino acid sequence VITWSGGITTYADSVKG (SEQ ID No.: 74) or VITVSGGITYYADSVKG (SEQ ID No.: 75), and CDR3 containing the amino acid DKHQSSWYDY (SEQ ID No.: 76) or DKHQSSFYDY (SEQ ID No.: 77) (the said ISVD is as disclosed in international patent applications WO2008071447, WO2017087587, and WO2017087589), the variant contains one, two, or three mutations in CDR3 as disclosed in WO2008071447, and one or more ISVDs have an effector-silencing antibody constant domain or an Fc domain, e.g., as disclosed herein It is excluded that an ISVD comprising the CDR in an embodiment fused to or connected to either the effector-silencing antibody constant domain or the Fc domain is not excluded by this proviso.

[0231] (f) Exemplary anti-PD-L1 antibody

[0232] An exemplary anti-PD-L1 antibody that may be used in the combination therapy of the present invention comprises any anti-PD-L1 antibody that inhibits PD-1 from binding to PD-L1, and further comprises a composition comprising the following anti-PD-L1 antibody and a pharmaceutically acceptable carrier. In a specific embodiment, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0233] In a specific embodiment, the anti-PD-L1 antibody is (i) V of atezolizumab H and V L Domain (here V H The domain is fused to or connected to an HC invariant domain or an effector-silence HC invariant domain, and V L(ii) the domain is fused to or linked to the LC kappa or lambda invariant domain, and (ii) V of avelumab H and V L Domain (here V H The domain is fused to or connected to an HC invariant domain or an effector-silence HC invariant domain, and V L The domain is fused to or linked to the LC kappa or lambda invariant domain), or (iii) V of durvalumab H and V L Domain (here V H The domain is fused to or connected to an HC invariant domain or an effector-silence HC invariant domain, and V L The domain includes (fused to or linked to the LC kappa or lambda invariant domain). Durvalumab V H The domain includes the amino acid sequence presented in sequence identification number: 103, and V L The domain contains the amino acid sequence presented in sequence identification number: 104. Avelumab V H The domain includes the amino acid sequence presented in sequence identification number: 105, and V L The domain contains the amino acid sequence presented in sequence identification number: 106. Atezolizumab V H The domain includes the amino acid sequence presented in sequence identification number: 107, and V L The domain comprises the amino acid sequence presented in sequence identification number: 108. In a specific 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 conservative or non-conservative mutations.

[0234] In a specific embodiment, anti-PD-L1 antibody V H The domain is currently a specific V HIt may be fused to or linked to an IgG1, IgG2, IgG3, or IgG4HC constant domain that is not linked to, or linked to an IgG1, IgG2, IgG3, or IgG4HC constant domain modified to include one or more mutations in the Fc domain that make the generated anti-PD-L1 antibody effector-silenced.

[0235] In certain embodiments, the HC constant domain is of the IgG1, IgG2, IgG3, or IgG4 isotype, which is modified to lack N-glycosylation of the asparagine (Asn) residue at position 297 of the HC constant domain by replacing the codon for Asn at position 297 of the nucleic acid molecule encoding the HC constant domain with another amino acid, e.g., a codon for Gln. Alternatively, the codon for Ser may be replaced with a codon for Pro, or the codon for Thr may be replaced with any codon other than the codon for Ser (e.g., N297A, N297G, or N297D). Alternatively, all three codons are modified. In further embodiments, this IgG modified to lack N-glycosylation at position 297 further comprises one or more additional mutations disclosed herein to remove detectable effector function. In certain embodiments, the HC constant domain is a human HC constant domain. In certain embodiments, the 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 conservative or non-conservative mutations.

[0236] In a specific embodiment, the present invention provides an anti-PD-L1 antibody comprising an IgG4HC constant domain modified to have an S228P substitution and, in addition to the S228P substitution, further include a P239G, D265A, or D265A / N297G amino acid substitution, wherein the positions are identified according to Eu numbering. In the specific embodiment above, the IgG4HC constant domain is a human HC constant domain. In a specific embodiment, the molecule 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.

[0237] In another embodiment, the anti-PD-L1 antibody may comprise a human IgG1 isotype, wherein the Fc domain of the HC constant domain is modified to be effector-silencing by substituting amino acids at positions 233 through 236 of IgG1 with the corresponding amino acids of human IgG2HC and amino acids at positions 327, 330 and 331 with the corresponding amino acids of human IgG4HC, wherein the positions are identified according to Eu numbering. In a specific 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.

[0238] In another embodiment, the Fc domain of the IgG1, IgG2, IgG3, or IgG4HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, wherein the polypeptide exhibits immeasurable or reduced binding to FcγRIIIA, FcγRIIA, and FcγRI compared to a polypeptide containing the wild-type IgG constant domain region as determined by a ViaCore assay. These and other substitutions are WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; and the literature [Sondermann et al. This is disclosed in Nature 406, 267-273 (20 Jul. 2000). In certain 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.

[0239] Tables 28-36 provide exemplary anti-PD-L1 antibodies that may be used in combination with anti-CTLA-4 antibodies as disclosed herein in a regimen for treating individuals with cancer. The invention also provides the antibodies and compositions presented in the table, with the exception of antibodies 25-9 and 31-8, each composition comprising the antibodies presented in the table and a pharmaceutically acceptable carrier, with the exception of antibodies 25-9 and 31-8. All HC amino acid substitution positions in Tables 28-36 are in accordance with the Eu numbering scheme.

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] (g) Exemplary anti-PD-L1 antibody fragment

[0246] An exemplary anti-PD-L1 antibody fragment that can be used in the combination therapy of the present invention comprises any anti-PD-L1 antibody fragment that binds to PD-L1 and inhibits PD-L1 from binding to PD-1, and further comprises the following anti-PD-L1 antibody fragments and compositions, each composition comprising the following anti-PD-L1 antibody fragment and a pharmaceutically acceptable carrier.

[0247] In a specific embodiment, the anti-PD-L1 antibody fragment is durvalumab V having the amino acid sequence presented in sequence identification number: 103. H and durvalumab V having the amino acid sequence presented in sequence identification number: 104 L It is an Fv or scFv containing.

[0248] In a specific embodiment, the anti-PD-L1 antibody fragment is durvalumab V having the amino acid sequence presented in sequence identification number: 103. H and durvalumab V having the amino acid sequence presented in sequence identification number: 104 H It is F(ab) containing .

[0249] In a specific embodiment, the anti-PD-L1 antibody fragment is durvalumab V having the amino acid sequence presented in sequence identification number: 103. H and durvalumab V having the amino acid sequence presented in sequence identification number: 104 H It is F(ab')2 containing .

[0250] In a specific embodiment, the anti-PD-L1 antibody fragment is an avelumab V having the amino acid sequence presented in sequence identification number: 105. H and Avelumab V having the amino acid sequence presented in Sequence Identification Number: 106 H It is an Fv or scFv containing .

[0251] In a specific embodiment, the anti-PD-L1 antibody fragment is an avelumab V having the amino acid sequence presented in sequence identification number: 105. H and Avelumab V having the amino acid sequence presented in Sequence Identification Number: 106 H It is F(ab) containing .

[0252] In a specific embodiment, the anti-PD-L1 antibody fragment is an avelumab V having the amino acid sequence presented in sequence identification number: 105. H and Avelumab V having the amino acid sequence presented in Sequence Identification Number: 106 H It is F(ab')2 containing .

[0253] In a specific embodiment, the anti-PD-L1 antibody fragment is atezolizumab V having the amino acid sequence presented in SEQ ID No. 107. H and atezolizumab V having the amino acid sequence presented in sequence identification number: 108 H It is an Fv or scFv containing .

[0254] In a specific embodiment, the anti-PD-L1 antibody fragment is atezolizumab V having the amino acid sequence presented in SEQ ID No. 107. H and atezolizumab V having the amino acid sequence presented in sequence identification number: 108 H It is F(ab) containing .

[0255] In a specific embodiment, the anti-PD-L1 antibody fragment is atezolizumab V having the amino acid sequence presented in SEQ ID No. 107. H and atezolizumab V having the amino acid sequence presented in sequence identification number: 108 H It is F(ab')2 containing .

[0256] In a specific embodiment, the anti-PD-L1 antibody fragment comprises one or more immunoglobulin monovariable domains (ISVDs), and each ISVD is a variable domain (V) of a camel-based heavy chain monoantibody. HH...including, provided that the ISVD does not include the claim-PD-L1 ISVD disclosed in international application WO2008071447 having sequence identification number: 394-399 or WO2009030285 (both of which are incorporated herein by reference), and that one or more ISVDs fused or linked to an effector-silencing antibody constant domain or Fc domain, e.g., any of the effector-silencing antibody constant domain or Fc domain disclosed herein are not excluded by this proviso.

[0257] (h) Exemplary combination therapy regimen

[0258] The present invention provides an anticancer therapy that combines the immunostimulatory effect of a PD-1 blocker with the antitumor effect of a CTLA-4 blocker, but without the cutaneous or intestinal irAEs typically observed with CTLA-4 blockers administered in combination with a PD-1 blocker. A feature of the present invention is that the CTLA-4 blocker lacks measurable binding to one or more FcRs as determined by the Viacor assay, or reduced binding to one or more FcRs compared to the same isotype wild-type antibody as measured by the Viacor assay. Therefore, the CTLA-4 blocker exhibits unmeasurable or reduced effector function, which enables the use of effector-silencing CTLA-4 blockers in combination therapy with PD-1 blockers at doses and durations of administration that cannot be used with CTLA-4 blockers exhibiting effector function. This feature distinguishes the CTLA-4 blocker of the present invention from currently available CTLA-4 blockers.

[0259] In a typical dosing regimen of the present invention, CTLA-4 blockers and PD-1 blockers may be co-administered to an individual at separate doses and in different forms. Generally, the CTLA-4 blocker of the present invention may be administered as a combination therapy with a PD-1 blocker at the same dose, dosing frequency, and duration of treatment currently approved by the U.S. FDA for ipilimumab / nivolumab combination therapy for at least specific indications. However, the combination therapy is not limited to specific indications approved by the U.S. FDA but may include any indications that may benefit from the combination therapy of the present invention. The currently approved dosage is 1 mg / kg of ipilimumab followed by the administration of nivolumab provided at a dose of 3 mg / kg. Subsequently, this dosage combination may be repeated every 3 weeks for 4 doses, and if necessary, administration of nivolumab may be continued every 2 weeks thereafter. However, in additional embodiments, the CTLA-4 blocker of the present invention may be administered in combination therapy at a dose greater than 1 mg / kg, e.g., at least 3 mg / kg. In yet another embodiment, the dose may be at least 10 mg / kg, and in yet another embodiment, the dose may be about 1 mg / kg to 10 mg / kg. In specific embodiments, the CTLA-4 blocker of the present invention may be administered at the same dosing frequency and duration of treatment as in approved ipilimumab / nivolumab combination therapy. In specific embodiments, the CTLA-4 blocker of the present invention may be administered at the same dosing frequency and duration of treatment as for nivolumab in approved ipilimumab / nivolumab combination therapy.

[0260] In a specific embodiment of the combination therapy, the CTLA-4 blocker is administered at a dose not based on the individual's body weight. Therefore, in a specific embodiment, the CTLA-4 binder may be administered at a dose of about 10 mg to 300 mg. In additional embodiments, the dose is selected from the group consisting of 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg.

[0261] In the combination therapy of the present invention, the PD-1 blocker may be administered at the same dose, frequency of administration, and duration of treatment as approved for the PD-1 blocker in monotherapy for a specific indication. The dose of the CTLA-4 blocker is as stated above, and the CTLA-4 blocker may be administered at the same frequency of administration and duration of treatment as stated above, or at the same frequency of administration and duration of treatment as for a specific PD-1 blocker paired with the CTLA-4 blocker.

[0262] Since specific dosages of currently marketed PD-1 blockers vary among them, in specific embodiments of the combination therapy of the present invention, the dosage, frequency of administration, and / or duration of treatment may be at least as approved by the U.S. FDA for the specific PD-1 blocker for the specific indication. For example, pembrolizumab is approved at a dose of 200 mg every 3 weeks as needed (up to 200 mg at 2 mg / kg every 3 weeks as needed for pediatric individuals (ages 2 to 18); nivolumab is approved at a dose of 3 mg / kg every 2 weeks; semiplimab-rwlc is approved at a dose of 350 mg every 3 weeks as needed; atezolizumab is approved at a dose of 1200 mg every 3 weeks as needed; and avelumab is approved at a dose of 10 mg / kg or 800 mg every 2 weeks as needed; Durvalumab was approved at a dose of 10 mg / kg every 2 weeks as needed.

[0263] 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 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 additional embodiments, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered at a frequency of every 3 weeks as needed. 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 at a frequency of every 6 weeks as needed at a dose greater than 250 mg, for example, about 400 mg.

[0264] 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 an anti-PD-1 antibody fragment may be administered at a frequency of every 2 to 3 weeks as needed.

[0265] PD-1 blockers may be administered at the dosage, frequency, and duration of treatment approved for currently marketed PD-1 blockers, at least in monotherapy; however, the actual dosage, frequency, and duration of treatment for any specific combination of the present invention may differ from those approved for PD-1 blocker monotherapy. Therefore, in a specific embodiment of the combination therapy of the present invention, the dosage, frequency, and duration of treatment of any specific PD-1 blocker in the combination therapy will be determined from clinical trials performed on the combination therapy.

[0266] In a specific embodiment of the combination therapy, the PD-1 blocker is nivolumab or an effector-silencing variant of nivolumab, which is administered intravenously to an individual at a dose of 3 mg / kg over 30 to 60 minutes every 2 to 3 weeks as needed, wherein each dose of the CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker. In a specific embodiment, nivolumab or an effector-silencing variant of nivolumab is administered intravenously to an individual over 30 minutes at an initial dose of 3 mg / kg, the CTLA-4 blocker is administered intravenously over 30 minutes on the same day every 3 weeks for 4 doses, and nivolumab is administered intravenously at a fixed dose of 240 mg over 30 minutes every 2 weeks or 480 mg over 30 minutes every 4 weeks.

[0267] In a specific embodiment, the PD-1 blocker is pembrolizumab or an effector-silencing variant of pembrolizumab, which is administered intravenously to adult subjects at a dose of 200 mg over 30 minutes every 3 weeks as needed, or to pediatric subjects at a dose of 2 mg / kg up to about 200 mg over 30 minutes every 3 weeks, wherein each dose of CTLA-4 blocker is followed by a dose of CTLA-4 blocker, wherein each dose of CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker, or for a duration shorter or longer than that of the PD-1 blocker.

[0268] In a specific embodiment, the PD-1 blocker is pembrolizumab or an effector-silencing variant of pembrolizumab, which is administered intravenously to an adult individual at a dose of 400 mg over 30 minutes every 6 weeks as needed, wherein each dose of CTLA-4 blocker is followed by a dose of CTLA-4 blocker after each treatment, wherein each dose of CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker.

[0269] In a specific embodiment of the combination therapy, the PD-1 blocker is semiplimab-rwlc or an effector-silencing variant of semiplimab-rwlc, which is administered intravenously to an individual at a dose of 350 mg over 30 minutes every 3 weeks as needed, wherein each dose of the CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker. In a specific embodiment, after semiplimab-rwlc or an effector-silencing variant of semiplimab-rwlc is administered intravenously to an individual at an initial dose of 350 mg over 30 minutes, the CTLA-4 blocker is administered over 30 minutes on the same day every 3 weeks as needed.

[0270] In a specific embodiment of the combination therapy, the PD-1 blocker is atezolizumab or an effector-silencing variant of atezolizumab, which is administered intravenously to an individual at a dose of 1200 mg over 60 minutes every 3 weeks as needed, wherein each dose of the CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker. In a specific embodiment, after the atezolizumab or effector-silencing variant of atezolizumab is administered intravenously to an individual at an initial dose of 1200 mg over 60 minutes, the CTLA-4 blocker is administered over 30 minutes on the same day every 3 weeks as needed.

[0271] In a specific embodiment of the combination therapy, the PD-1 blocker is avelumab or an effector-silencing variant of avelumab, which is administered intravenously to an individual at a dose of 10 mg / kg or 800 mg over 60 minutes every 2 weeks as needed, wherein each dose of the CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker. In a specific embodiment, after the avelumab or an effector-silencing variant of avelumab is administered intravenously to an individual at an initial dose of 10 mg / kg or 800 mg over 60 minutes, the CTLA-4 blocker is administered over 30 minutes on the same day every 2 weeks as needed.

[0272] In a specific embodiment of the combination therapy, the PD-1 blocker is durvalumab or an effector-silencing variant of durvalumab, which is administered intravenously to an individual at a dose of 10 mg / kg over 60 minutes every 2 weeks as needed, wherein each dose of the CTLA-4 blocker is administered intravenously after administration of the PD-1 blocker for a duration of treatment equal to that of the PD-1 blocker or for a duration shorter or longer than that of the PD-1 blocker. In a specific embodiment, after the durvalumab or effector-silencing variant of durvalumab is administered intravenously to an individual at an initial dose of 10 mg / kg over 60 minutes, the CTLA-4 blocker is administered over 30 minutes on the same day every 2 weeks as needed.

[0273] Currently approved CTLA-4 blockers and PD-1 blockers are provided in formulations at concentrations that allow intravenous administration to an individual over a time frame of 30 to 60 minutes, but the combination therapy of the present invention considers an embodiment in which the CTLA-4 blocker and / or PD-1 blocker are each provided in formulations at concentrations that allow them to be administered separately to an individual as a single injection. Being able to provide at least one of the two blockers as a single injection will significantly reduce the time required to administer both blockers to an individual.

[0274] 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 may be achieved by providing the CTLA-4 and PD-1 blockers in separate formulations and administering each formulation to the subject simultaneously by separate IVs or by mixing them before administering the mixture to the subject by IV, or by administering each formulation to the subject by separate injections. Co-administration may also be achieved by providing the CTLA-4 and PD-1 blockers in a single formulation, which is subsequently administered to the subject by a single IV or a single injection.

[0275] (i) Combination therapy

[0276] The combination therapy of the present invention may be used for the treatment of any proliferative disease, particularly for the treatment of cancer. In certain embodiments, the combination therapy of the present invention may 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 lymphoma, renal cancer, Hodgkin 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.

[0277] In another embodiment, the combination therapy of the present invention may be used to treat pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, cancer of the uterus or endometrium, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, cancer of the small intestine or appendix, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

[0278] PD-1 blockers currently on the market have been approved by the U.S. FDA to treat at least one cancer 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 cutaneous squamous carcinoma. Therefore, the combination therapy of the present invention can be used to treat at least one cancer 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 cutaneous squamous carcinoma.

[0279] (j) Combination therapy combined with chemotherapy

[0280] The combination therapy of the present invention may be administered to an individual having cancer in combination with chemotherapy. The individual may receive chemotherapy simultaneously with receiving the combination therapy of the present invention. The individual may receive the combination therapy of the present invention after completing chemotherapy. The individual may receive chemotherapy after completing the combination therapy. The combination therapy of the present invention may also be administered to an individual having recurrent or metastatic cancer with disease progression or recurrence who is receiving or has completed chemotherapy.

[0281] Chemotherapy may include chemotherapy agents selected from the group consisting of the following:

[0282] (i) alkylating agents including, but not limited to, difunctional alkylating agents, cyclophosphamide, mechlorethamine, chlorambucil and melphalan;

[0283] (ii) monofunctional alkylating agents including, but not limited to, dacarbazine, nitrosourea, and temozolomide (oral dacarbazine);

[0284] (iii) anthracyclines including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and valubicin;

[0285] (iv) Cytoskeletal disruptors (taxanes) including but not limited to paclitaxel, docetaxel, abraxane and taxotere;

[0286] (v) Epotillons including, but not limited to, ixabephylon and utidelon;

[0287] (vi) histone deacetylase inhibitors including, but not limited to, vorinostat and romidepsin;

[0288] (vii) Inhibitors of topoisomerase i, including but not limited to irinotecan and topotecan;

[0289] (viii) Inhibitors of topoisomerase ii, including but not limited to etoposide, tenifoside and taflufoside;

[0290] (ix) Kinase inhibitors including, but not limited to, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib and bismodezyb;

[0291] (x) nucleotide analogs and precursor analogs including, but not limited to, azacitidine, azathioprine, fluoropyrimidine (e.g., capecitabine, carmofur, doxyfluridine, fluorouracil, and tegafur), cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly thioguanine);

[0292] (xi) peptide antibiotics including, but not limited to, bleomycin and actinomycin; platinum-based agents including, but not limited to, carboplatin, cisplatin and oxaliplatin;

[0293] (xii) retinoids including, but not limited to, tretinoin, alitretinoin, and bexarotene; and

[0294] (xiii) Vinca alkaloids and derivatives including but not limited to vinblastine, vincristine, vindesin and vinorelbine.

[0295] The selection of dosage for chemotherapy agents depends on several factors, including the serum or tissue turnover of the subject, symptom level, immunogenicity of the subject, and the accessibility of target cells, tissues, or organs in the subject to be treated. The dosage of additional therapeutic agents must be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of each additional therapeutic agent will depend in part on the specific agent, the severity of the cancer to be treated, and patient characteristics. Guidelines for the appropriate dosage selection of antibodies, cytokines, and small molecules are available. For example, the literature [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.See [343:1594-1602]; [Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed)]; [Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)]. The determination of an appropriate dose regimen may be made by a clinician using parameters or factors known or suspected in the relevant technical field that affect or are expected to affect treatment, for example, and will depend on the individual's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated, and biomarkers of response to one or more therapeutic agents in combination therapy.

[0296] For example, pembrolizumab is currently approved by the U.S. FDA for combination therapy for the treatment of non-small cell lung cancer (NSCLC) including (i) pembrolizumab with pemetrexid and platinum chemotherapy or carboplatin and paclitaxel or nab-paclitaxel; and (ii) pembrolizumab with platinum-containing chemotherapy for the treatment of head and neck squamous cell carcinoma (HNSCC), and atezolizumab is currently approved for combination therapy for the treatment of NSCLC including bevacizumab (an anti-VEGF-A antibody marketed as Avastin), paclitaxel, and carboplatin.

[0297] Therefore, the present invention considers embodiments of the combination therapy of the present invention further comprising a chemotherapy step comprising platinum-containing chemotherapy, pemetrexid and platinum chemotherapy, or carboplatin and paclitaxel or nab-paclitaxel. In certain embodiments, the combination therapy with the chemotherapy step may be used to treat at least NSCLC and HNSCC.

[0298] Additionally, a combination therapy combined with a chemotherapy step may be used for the treatment of any proliferative disease, particularly cancer. In certain embodiments, the combination therapy of the present invention may 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 lymphoma, renal cancer, Hodgkin 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.

[0299] In another embodiment, a combination therapy combined with an additional chemotherapy step may be used to treat pancreatic cancer, bronchial cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, cancer of the small intestine or appendix, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

[0300] In certain embodiments, combination therapy with a chemotherapy step may 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 cutaneous squamous carcinoma.

[0301] The following examples are intended to facilitate further understanding of the present invention.

[0302] Example 1

[0303] Fc-function is required for the induction of intestinal inflammation irAE.

[0304] Intestinal inflammatory irAEs have been observed in cancer patients during anti-CTLA-4 antibody monotherapy or immunotherapy combined with anti-PD-1 antibodies. Preclinical studies of CTLA-4-deficient mice in constitutive and conditional knockout models demonstrated the development of severe immune-mediated inflammatory diseases across multiple organs. However, treatment of synonymous tumor models using surrogate anti-CTLA-4 antibodies has not been reported to induce apparent irAEs predicting the toxicity observed in cancer patients. Similarly, histopathological evaluations for intestinal inflammation were assessed in CT26 synonymous or xenograph models (mice inoculated with CT26 colon carcinoma cell lines). CT26 tumor-bearing mice treated with the Fc-qualified anti-mouse CTLA-4 mAb 9D9-mIgG2a (α-CTLA-4 or α-CTLA4) (q4x4) resulted in minimal granulocyte infiltrations (Grade 1 of Grade 5) observed in the lamina propria intestinal tissue. Granulocyte infiltrations were not observed in cohorts treated with the Fc-mutant anti-mCTLA-4 mAb 9D9-mIgG1-D265A (α-CTLA-4 (D265A)), the Fc-free anti-mCTLA-4 ISVD F894 (CTLA-4 Nab), or isotypes. However, no associated ulcers or other tissue damage were observed.

[0305] The α-CTLA-4 (D265A) Fc-mutant lacks measurable affinity for the Fcγ receptor (Nimmerjahn et al., Immunity, 23: 41-51 (2005)) and, consequently, lacks Fc-effector function. In mice, the antitumor efficacy of anti-mouse CTLA4 mAb monotherapy depends on the antibody's ability to mediate intratumor regulatory T cell depletion through Fc-effector function (Selby et al., op. cit). As such, neither CTLA-4 Nab nor α-CTLA-4 (D265A) were expected to have monotherapy antitumor benefits.

[0306] Minimal histopathological findings prompted the evaluation of gene expression profiles as a potentially more sensitive means for detecting markers of inflammatory cell activation in the intestine. The inventors utilized a PCR gene-expression panel previously developed by the inventors for the proteomics and expression profiling of genes associated with intestinal inflammation in stool samples and biopsies from patients and preclinical models of inflammatory bowel disease (IBD) (Cayatte, C, et al., Clinical and Translational Gastroenterology, 3: e10 (2012)).

[0307] Gene expression profiles were measured in small and colon tissue samples at various times after the initiation of treatment with α-CTLA-4, and the role of Fc-function in induced intestinal inflammation was specifically evaluated in comparison to α-CTLA-4 (D265A). As illustrated in Figure 1a, the expression of numerous intestinal inflammation 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). The results suggested that the expression of intestinal inflammation pathways can be detected by gene expression in small and colon tissues in a subclinical setting.

[0308] The upregulation of genes associated with intestinal inflammation allowed for the evaluation of the effects of continued treatment on progression to clinical enterocolitis, as observed in ipilimumab-treated patients after 6 to 7 weeks or more of treatment (Samaan et al., Nat. Rev. Gastroenterol. Hepatol. 15: 222-234 (2018)). To evaluate 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 and α-CTLA-4 (D265A) twice weekly. Two groups of mice were treated with Fc-eligible α-CTLA-4 (one group of mice with CT26 tumors and one group of naive BALB / c mice without tumors) to evaluate the potential contribution from the induction of tumor immunity to tumor growth and the induction of intestinal inflammation. To monitor progression to enterocolitis, weight and physical condition scores were evaluated twice a week throughout the entire continuous treatment.

[0309] Body weight continued to increase until day 50 in mice treated with the isotype antibody and α-CTLA-4 (D265A). In contrast, mice treated with α-CTLA-4 showed a decrease in mean body weight after about 35 to 40 days (Fig. 1b). Intestinal permeability increased in both α-CTLA-4 treatment groups as assessed in FITC-dextran tube-fed mice at days 49 and 50, but did not increase in mice treated with α-CTLA-4 (D265A) (Fig. 1c). Histological evidence of inflammation in the proximal small intestine and colon, as assessed by a pathologist (LA), indicated progression to moderate and severe enterocolitis in the α-CTLA-4 treatment groups (Figs. 1d and 1e), with extensive immune infiltration, mucosal thickening, and loss of goblet cells. In contrast, enterocolitis was not observed in α-CTLA-4 (D265A)-treated mice, providing evidence that Fc-function is required for CTLA-4 blockade-induced enterocolitis. In particular, tumor growth and anti-tumor responses were not required for α-CTLA-4-induced intestinal inflammation.

[0310] Mouse IgG with high FcγR affinity 2a - In experiments comparing chimeric antibodies with detectable FcγR-absent mutant IgG1 chimeric antibodies, it was previously reported that anti-CTLA-4 antibodies with strong FcγR function are required for a strong monotherapy antitumor response (Selby et al., Cancer Immunol. Res. 1: 32-42 (2013)). In tumors, T reg Specific depletion of is surface-related to mouse IgG 2a (mIgG 2aIt plays a key role in the potent monotherapy observed with anti-CTLA-4 antibodies in the backbone (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 interactions between CTLA-4 and CTLA-4 antagonists on ligands CD80 and CD86 is T reg It was suggested that strong combination antitumor efficacy could be provided without the requirement for TIL depletion. An ISVD specific to mouse CTLA-4 (mCTLA-4) capable of inhibiting binding to CD80 and CD86 was developed using a single-domain VHH antibody fragment derived from a heavy-chain-only camelid antibody. These small 15 kDa proteins lack an Fc domain and therefore do not bind to FcγR. For comparison with a fully Fc-functional antibody, the inventors utilized α-CTLA-4, which was previously presented as having strong monotherapy antitumor efficacy (see reference). To control the potential effects of different CTLA-4 epitope bindings, the inventors utilized α-CTLA-4 (D265A), which lacks detectable affinity for FcγR, to control the requirement for strong FcγR function for antitumor efficacy and tolerability.

[0311] Two anti-CTLA-4 V connected by a 35GS-linker HH Anti-human albumin V as a domain and half-life extension (HLE) subunit HH CTLA-4 nAb composed of domains (see Sequence Identification Number: 61). Anti-CTLA-4 V HHIt scored 96% inhibition for both CD80 and CD86 compared to α-CTLA-4 used as a reference antagonist (100%) (Figs. 2a and 2b). CTLA-4 nAb was further compared with α-CTLA-4 and effector-silenced α-CTLA-4 (D265A) in an in vitro MLR-based bioassay for its ability to increase proliferation (Fig. 2c), IFNγ (Fig. 2d), and IL-2 response (Fig. 2e).

[0312] Fc-function for CTLA-4 blockade is not required in PD-1 combination immunotherapy.

[0313] 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 a synonymic CT26 colorectal carcinoma tumor model (Fig. 3a-3e) that responded only moderately to anti-PD-1 monotherapy. No antitumor activity was observed in the monotherapy cohort treated with CTLA-4 nAb or α-CTLA-4 (D265A), which was comparable to the isotype control group (Group 1 in Fig. 3a). Strong antitumor monotherapy activity was observed in mice treated with α-CTLA-4, which was consistent with previous reports demonstrating the requirement for the Fc-function of anti-CTLA-4 antibodies for antitumor monotherapy efficacy in mouse synonymic tumor models (Simpson et al., J. Exp. Med. 210: 1695-710 (2013)) (Group 2 in Fig. 3a), while treatment with α-PD-1 alone provided low to moderate antitumor growth inhibition compared to α-CTLA-4. However, combination therapy with α-PD-1 and CTLA-4 nAb or α-CTLA-4 (D265A) provided strong antitumor benefits comparable to those observed with α-CTLA-4 alone or α-CTLA-4 combined with α-PD-1 (Group 2 in Fig. 3a). Since CTLA-4 nAb and α-CTLA-4 (D265A) bind to separate epitopes on CTLA-4, the effect is not specific to the epitope. Because similar antitumor responses were observed in α-PD-1 combination therapy among CTLA-4 nAb, α-CTLA-4 (D265A), and α-CTLA-4, additional evidence for a potent combination benefit independent of Fc-function was evident. Figure 3d presents the individual results for each of the 10 mouse treatments summarized in Figure 3a.Expansion of CD8 T cells and an increased CD8 / Treg ratio were observed in mice treated with α-CTLA-4 and mice treated with the combination of α-CTLA-4 (D265S) plus α-PD-1 compared to mice treated with α-CTLA-4 (D265S) alone (Fig. 3b). These results indicate that the Fc-deficient anti-CTLA-4 antagonist combined with the anti-PD-1 antagonist provided superior antitumor efficacy compared to that achievable with anti-PD-1 antagonist monotherapy, and provided antitumor efficacy in a combination similar to that of the Fc-functional anti-CTLA-4 antibody in monotherapy.

[0314] To elucidate potential complementary mechanisms associated with strong combination activity, the inventors investigated the regulation of immune response genes associated with effective cancer immunotherapy in tumors 9 days after the initiation of treatment. PCR-expression profiling of tumors from mice treated with α-CTLA-4 revealed strong upregulation of numerous genes associated with effective immunotherapy, including IFNγ, IFN-responsive genes, chemokines, pro-inflammatory cytokines, and MHC (Fig. 3c). Only slight upregulation was observed in tumors from mice treated with α-CTLA-4 (D265A), indicating that the strong upregulation observed in α-CTLA-4 treated tumors is at least partially dependent on Fc-function. Slight responses were also observed in tumors from mice treated with CTLA-4 nAb and α-PD-1. In contrast, strong upregulation of tumor immune response genes was observed in combination therapy cohorts treated with CTLA-4 nAb or α-CTLA-4 (D265A) + α-PD-1. Figure 3d suggests that CTLA-4 nAb or α-CTLA-4 (D265A) does not have antitumor activity in the absence of PD-1 blockade. These data support the hypothesis that a complementary mechanism to pure CTLA-4 blockade and PD-1 blockade can provide a strong combinational advantage in an Fc-independent manner.

[0315] Fc-nonfunctional CTLA-4 blockade combined with anti-PD-1 provides an excellent therapeutic index.

[0316] The outstanding feature of immune checkpoint blockade is the clinically validated combination benefit of anti-PD-1 and anti-CTLA-4 antibodies, which generates superior clinical efficacy compared to targeting the checkpoint pathway alone. However, immuno-related toxicity (irAE) associated with CTLA-4 blockade combination therapy with anti-PD-1 was associated with 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 required combination with α-PD-1 to induce strong anti-tumor immunity. To control the potential effect of strong tumor immunity on the induction of intestinal inflammation, the inventors examined the intestinal inflammation expression profiles in mice administered combination therapy with α-PD-1 and α-CTLA, α-CTLA (D265A), or CTLA-4 nAb after 5 treatments on day 18 after the initiation of treatment (Fig. 4d).

[0317] To evaluate the relative effects of CTLA-4 blockade and Fc-function on intestinal inflammation, naive BALB / c mice were administered α-CTLA, α-CTLA (D265A), Fc-free CTLA4 nAb, α-PD-1, or combinations of various anti-mCTLA4 antagonists and α-PD-1 twice weekly. Body weight and body condition scores were assessed twice weekly throughout the study. Body weight of mice in all groups increased until approximately day 20 (Fig. 4a). Body weight continued to increase until day 50 in mice administered isotypes, α-CTLA (D265A), CTLA4 nAb, or α-PD-1 or combinations thereof. Mice administered α-CTLA showed a decrease in mean body weight after approximately day 30 and were found to decrease to near pre-treatment levels by day 50. The administration of α-CTLA combined with α-PD-1 induced a more rapid decrease in average body weight to below pre-treatment levels from day 20 to day 50. Specifically, in mice administered α-CTLA, the body condition score for 2 out of 8 mice dropped to 2 (under conditioning) starting on day 42, whereas in mice treated with α-CTLA combined with α-PD-1, it started on day 28. These cohorts exhibited glossy, coarse fur, and a swollen abdomen was observed in these cohorts.

[0318] Inflammation analysis was scheduled 7 weeks after administration, and mice treated with α-CTLA exhibited body weight loss over a period of time, which worsened when administered in combination with α-PD-1 (Group B in Fig. 4a). In contrast, neither the effector-silencing CTLA-4 blocker nor α-PD-1 showed any significant body weight loss during this period compared to the isotype control group (Group A in Fig. 4b).

[0319] All mice from the combination therapy group and 4 mice from the isotype control and α-PD-1 therapy groups were euthanized on day 50 for tissue collection. The remaining 4 mice from the isotype control and α-PD-1 therapy groups and all mice from the single-agent therapy group were euthanized on day 54 for tissue collection. At the time of necropsy, the proximal small intestine and colon were resected for RT-qPCR to determine the expression of inflammatory genes and to evaluate inflammation by histopathology.

[0320] Heatmaps of gene expression in the proximal small intestine from each treatment group relative to the isotype control group are presented in (Fig. 4d). Administration of α-CTLA was sufficient to induce upregulation of inflammatory genes in the jejunum (Fig. 4d) and colon (Fig. 4e). The combination of α-PD-1 and α-CTLA induced much stronger upregulation of inflammatory genes than α-CTLA monotherapy. In contrast, administration of CTLA-4 nAb in combination with α-PD-1 induced little to no intestinal inflammatory gene expression, and only slight 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 gastrointestinal feeding was significantly increased in mice treated with α-CTLA and mice treated with the combination of α-CTLA and α-PD-1.

[0321] The severity of inflammation in the proximal small intestine was scored by histological evaluation of enteritis in the proximal jejunum on day 50. Histopathological evaluation revealed that administration of α-CTLA resulted in mild to severe inflammation in most mice. In the cohort treated with a combination of α-CTLA and α-PD-1, continuous treatment induced moderate to very severe inflammation in all mice (Fig. 4b). Mice with very severe enteritis exhibited jejunitis, diffuse neutrophil lesions with a moderate number of mast cells, and neuronal degeneration of the Meissner plexus. In contrast, administration of CTLA-4 nAb or α-CTLA (D265A) did not induce inflammation in histopathological evaluation. Administration of CTLA-4 nAb in combination with α-PD-1 resulted in no inflammation or minimal to mild inflammation in several mice. Administration of α-CTLA (D265A) in combination with α-PD-1 induced mild inflammation in only 1 out of 8 mice. Representative micrographs show the relative levels of inflammation in each treatment group (Fig. 4c).

[0322] As shown in Figures 5a-5c, the α-CTLA-4 Fc effector function induces skin inflammation (Figure 5a) but not systemic inflammation, where no detectable inflammation was found in the kidneys, liver, or lungs (Figure 5c). The absolute number of IL-17-producing T cells, Foxp3+ Treg cells, and neutrophils in the ear skin was measured by flow cytometry. As shown in Figure 5b, elevated levels of IL-17-producing T cells, Foxp3+ Treg cells, and neutrophils were present in the ear skin of mice treated with α-CTLA-4, but not in mice not treated with α-CTLA-4 (D265A). Taken together, these data support the key role of the Fc-effector function in the induction of intestinal inflammation by anti-mouse CTLA-4 antibodies with effector function. Fc-effector function contributed to anti-mouse α-CTLA-induced intestinal inflammation in the BALB / c mouse model of enterocolitis, whereas intestinal inflammation was mild or absent in mice treated with CTLA-4 nAb or mice treated with α-CTLA (D265A).

[0323] In summary, two attributes were associated with the induction of intestinal inflammation by α-CTLA-4 in the CT26 tumor model. First, CTLA-4 specificity was required because the Fc-functional isotype control did not induce inflammation-associated gene expression. However, blocking CTLA-4 binding to CD80 / CD86 ligands was insufficient to induce the upregulation of inflammatory genes in the intestines of mice treated with CTLA-4 nAb and α-CTLA-4 (D265A). Therefore, IgG of α-CTLA-4 2a The strong Fc-functional capacity present in the isoforms was required for the induction of intestinal inflammation.

[0324] The activation of intestinal inflammation is T reg Independent of the depletion of T eff It is initiated by cell activation.

[0325] The mechanism of action (MOA) for anti-CTLA-4 mediated antitumor immunity is theoretically T regulation (T reg ) not only cells but also T effectors (T eff It is mediated by pharmacodynamic (PD) effects on cell populations (CTLs, TH1 cells, etc.). T within the tumor microenvironment (TILs) reg Cell depletion is a dominant MOA for anti-CTLA-4 antibodies in murine synonyms tumor models (Simpson et al., op. cit.). Furthermore, Fc-FcγR co-binding by anti-CTLA-4 mAbs modulates T cell receptor (TCR) and CD28 signaling, thereby affecting T reg It resulted in enhanced T cell activation independently of depletion (Waight et al., Cancer Cell, 33: 1033-1047 (2018)).

[0326] T reg To characterize the differential effects of α-CTLA-4 from CTLA-4 nAbs on T effector cells, flow cytometry was performed on T cell populations from tumors (TILs), the lamina propria of the colon, blood, and spleen 20 hours after subcutaneous administration. Since it does not perform cross-blocking that enables staining of drug-conjugated CTLA-4, T cells from mice treated with α-CTLA-4 or α-CTLA-4 (D265A) were analyzed using the anti-CTLA-4 mAb clone UC10-4B9 (ThermoFisher). reg We were able to measure the CTLA-4 expression levels. As previously reported in the literature (Selby et al. op. cit., Simpson et al. op. cit.), the inventors [were able to] measure T in the spleen of CT26 tumor-bearing mice. reg (CTLA-4 lo ) and tumor microenvironment (T reg hi Differential expression of CTLA-4 was observed in ). T in PBMCs regCTLA-4 lo-mid It indicated the bimodal level of. Interestingly, T from the lamina propria of the colon reg CTLA-4 mid-hi It showed the bimodal level of CTLA-4 in the colon. hi T reg is T reg It showed levels similar to the TIL population. Differential expression levels of CTLA-4 across various T cell populations influence ADCC-mediated depletion capacity due to receptor density-dependent apoptosis mechanisms. reg The population normally expresses higher levels of CTLA-4, but T in the tumor environment reg is at a much higher level than that found in the spleen (MFI= 2,400) (T reg It expresses 3.3 times higher (MFI = 8,100) in the isotype control of TILs. lamina propria T from the colon expressing higher CTLA-4 levels. reg (CTLA-4 hi Mode MFI = 10,000) is T for relative expression levels using flow cytometry reg It was similar to TIL (Fig. 6a). As exemplified in Figs. 6b-6d, T reg Significant depletion of was limited to TILs from the tumor microenvironment of mice treated with α-CTLA-4, which has the highest density of CTLA-4 expression. Treatment with the Fc-deficient Fc-mutant α-CTLA-4 (D265A) T reg It did not cause depletion.

[0327] Based solely on the assumption that cells expressing higher levels of CTLA-4 are susceptible to depletion by α-CTLA, the inventors T reg CTLA-4 of the eigenplate similar to the TIL population hi T reg It was predicted that it would be depleted. Surprisingly, T isolated from tumors of α-CTLA-4 treated mice regOnly TILs were found to be depleted. TILs derived from the lamina propria of the colon from α-CTLA-4 treated mice reg It was found that it was not depleted. T derived from the lamina propria of the colon from α-CTLA-4 treated mice reg No detectable depletion of was observed, which suggests that the induction of intestinal inflammation is T in the intestinal mucosa reg It suggests that it was not initiated by the loss of.

[0328] T affecting inhibitory function that may contribute to α-CTLA-4-induced intestinal inflammation reg Possible phenotypic changes were investigated. Colon lamina propria (LP) T from MC38 tumor-transplanted FoxP3 GLD reporter mice. reg ul T reg They were classified for PCR expression profiling of function-associated genes (Fig. 7a). However, compared to α-CTLA-4 (D265A) treated mice, LP T from α-CTLA reg No significant differences in gene expression were observed in the cells. T reg The potential effect of Fc-function on CD45RB in colitis hi Further investigation was conducted using a T cell delivery model. CD45RB hi T using T cells reg Passive delivery of cells protected mice from the development of colitis (Figs. 7b-7c). CD45RB with α-CTLA-4 hi T cells and T reg Treatment of mice co-administered with cells is T reg It resulted in loss of protection and the development of colitis. In contrast, no loss of protection was observed in mice treated with CTLA-4 nAb (Figs. 7b-7c). Flow cytometry-sorted colon Foxp3+ T from mice 24 hours after treatment regThe gene expression profile from cells showed significant upregulation of gene expression in mice treated with α-CTLA-4, which was compared to the gene expression observed in cells obtained from mice treated with CTLA-4 nAb or isotype controls, with CD45RB high These results are similar to those observed in. Collectively, these results are Fc-mediated T reg Although depletion is not essential for the induction of intestinal inflammation by α-CTLA-4, T in response to intestinal inflammation reg It suggests that the control function of can be adjusted.

[0329] Limited expression of CTLA-4 and CD28 on T cells, and CD80, CD86, and FcγR on antigen-presenting cells is T reg It may play a key role in the Fc-functional α-CTLA-4 nAb activation of T cells independently of depletion (Waight et al., Cancer Cell, 33: 1033-1047, 2018). While Fc-enhanced activation is beneficial in tumors, it may contribute to unintended irAEs in intestinal tissues. We investigated the potential contribution of Fc-function to immune effector cell activation in intestinal tissues. Flow cytometry analysis of CD16 / 32 expression on macrophages from both tumors and the colon revealed significantly higher levels of FcR on antigen-presenting cells compared to splenic macrophages (Figs. 8a & 8b). Additionally, CD45 in tumors and the colonic lamina propria + CD11b + F4 / 80 + The proportion of macrophages was substantially higher than in the spleen (Fig. 8c). Fc-function was required for the activation of IL1β, TNFα, and IFNγ cytokine responses in intestinal tissues and was evident 10 days after the initiation of treatment, long before there was clear evidence of intestinal inflammation suggesting a significant role in irAE induction (Fig. 8c).

[0330] Cytokine responses in CD4 T cells isolated from the colonic lamina propria of α-CTLA-4 treated mice after 1 month of treatment showed a higher proportion of IL-17, TNFα, and IFNγ-producing cells associated with Fc-mediated intestinal inflammation (Fig. 8e). Additionally, an Fc-dependent increase in neutrophils was induced in α-CTLA-4 treated mice as well (Fig. 8e). Collectively, the results indicate that CTLA-4 blockade-associated intestinal inflammation is induced by Fc-mediated activation of effector cells and amplified by FcγR-antibody enhancement of cell cross-linking between T cells and APCs, which leads to the stimulation of inflammatory cytokine responses as exemplified in Fig. 11. Fc-mediated induction of intestinal inflammation is T reg It can be induced by effector T cells independently of depletion.

[0331] argument

[0332] Several recent reports have supported the key role of Fc-function in CTLA-4 blockade monotherapy in winter mouse cancer immunotherapy tumor models (International Patent Application WO 2014 / 089113; literature [Selby et al., Cancer Immunol. Res. 32-42 (2013)]; [Vargas et al., Cancer Cell, 33: 649-663 (2018)]). Ingram et al. reported in literature [Proc. Natl. Acad. Sci. USA 115: 3912-3917, (2018)] that fusing the Fc domain to ISVD is necessary to provide anti-CTLA-4 ISVD with antitumor efficacy. Indeed, the inventors observed a similar lack of efficacy when treating tumors with CTLA-4 nAb or α-CTLA-4 (D265A) as monotherapy. Tumor-infiltrating T expressing higher cell surface CTLA-4 levels regSpecific depletion of has been proven 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).

[0333] Induction of enterocolitis by α-CTLA-4 is T present in the lamina propria. reg It was not associated with detectable depletion. The inventors' study expands the potential for CTLA-4 blockade in anticancer therapy by demonstrating strong antitumor activity without inducing strong intestinal inflammation when combined with anti-PD-1 antibodies. Fc-function was not required to achieve the combination benefits of CTLA-4 and PD-1 blockade. Combination therapy containing α-PD-1 and CTLA-4 nAb or α-CTLA-4 (D265A) induced similar activation of IFNγ-associated immune response genes in tumors as induced by α-CTLA-4 possessing strong Fc-function. In contrast, strong intestinal inflammation progressing to enterocolitis was primarily observed in mice treated with α-CTLA-4 and was increased when combined with the anti-PD-1 antibody mDX400. These results indicate that simple blockade of CTLA-4, which promotes CD28 activation, is sufficient to increase the antitumor response of depleted T cells when combined with PD-1 blockade.

[0334] In the literature [Science 355: 1423-1427 (2017)], a previous report by Kamphorst et al. demonstrated that the PD-1 blockade structure of depleted CD8 T cells requires CD28 co-stimulation of TCR activation. Furthermore, in the literature [Science 355: 1428-1433 (2017)], a companion report by Hui et al. demonstrated that the co-receptor CD28 is more strongly preferred than the TCR as a target for dephosphorylation by PD-1-mobilized Shp2 phosphatase, and that CD28 is preferentially dephosphorylated. The results of the inventors suggest that complementary activation of the TCR co-receptor CD28 and blockade of PD-1-mediated dephosphorylation of CD28, mediated by simple blockade of CTLA-4 by an Fc-nonfunctioning anti-CTLA-4 antibody, may be sufficient to achieve combination benefits for cancer immunotherapy. The advantage of simple combination blockade without Fc-mediated enhanced activation via Fc-FcγR bridging is that it can allow for a larger therapeutic index, enabling a higher dose range and longer treatment times. This advantage can also facilitate additional combinations with standard chemotherapy regimens due to a lower intestinal inflammatory irAE risk profile.

[0335] Experimental Procedure

[0336] mouse

[0337] Wild-type C57BL / 6J mice were obtained from Jackson Laboratories. Wild-type Balb / c and CB17-SCID mice were obtained from Taconic. B6.Foxp3GDL (GFP-DTR-luciferase) mice were generated and maintained under specific pathogen-free conditions and housed in air-filtered microisolators at the Merck Research Laboratories (MRL) animal facility in Palo Alto, California, USA. All animal procedures were approved by MRL’s Institutional Animal Care and Use Committee in accordance with the guidelines of the Association for Evaluation and Accreditation of Laboratory Animal Care.

[0338] Tumor Tasks and Treatment

[0339] For synchronized tumor experiments, CT26, MC38, and MB49 tumor models were used. 3 x 10⁶ tumors were implanted in the flanks of 8- to 12-week-old Balb / c or C57BL / 6J mice. 5 Canine CT26 cells were injected subcutaneously (sc). Tumor diameter was measured using electronic calipers, and tumor volume was calculated by length × width × width × 1 / 2. Treatment was initiated when the tumor reached approximately 100 mm³. Mice were treated subcutaneously (sc) twice a week at 10 mg / kg with α-CTLA-4, α-CTLA-4 (D265A), mouse anti-IgG1-D265A antibody isotype control, mouse anti-IgG2a antibody isotype control, and α-PD-1 antibody. Mice were treated sc twice a week at 30 mg / kg with CTLA-4 nAb or ISVD controls.

[0340] α-CTLA-4 includes HC having the amino acid sequence of sequence identification number: 58 and LC having the amino acid sequence of sequence identification number: 59.

[0341] α-CTLA-4 (D265A) includes HC having the amino acid sequence of sequence identification number: 60 and LC having the amino acid sequence of sequence identification number: 60.

[0342] α-PD-1 includes HC having the amino acid sequence of sequence identification number: 63 and LC having the amino acid sequence of sequence identification number: 64.

[0343] CTLA-4 nAb contains the amino acid sequence presented in sequence identification number: 61.

[0344] Anti-PD-1 ISVD F037 (PD-1 nAb) contains the amino acid sequence presented in sequence identification number: 62.

[0345] Induction of colitis and skin inflammation

[0346] Naive 8- to 12-week-old Balb / c mice were sc-treated at 20 mg / kg twice weekly for 8 weeks with antibodies including α-CTLA-4, α-CTLA-4 (D265A), mouse anti-IgG1-D265A isotype control, mouse anti-IgG2a isotype control, and α-CTLA-4 nAb. Mice were sc-treated at 30 mg / kg twice weekly with CTLA-4 nAb or ISVD control. On day 55, plasma was collected for ELISA and Luminex assays. Organs were collected and treated as follows: 1) fixed in 10% neutral buffered formalin and paraffin-embedded tissue sections stained with H&E for histopathology evaluation; 2) rapidly frozen in liquid nitrogen for further RNA extraction; or 3) placed in HBSS for cell isolation.

[0347] T cell-induced colitis

[0348] Splenocytes from Balb / c mice were processed and purified for CD4 using magnetic bead separation (stem cell technology). TCRb+ CD4+ CD25- CD45RB highT cells (CD45RB-high T cells) and TCRb+ CD4+ CD25+ CD45RB-low (T reg The cells were classified into FACS Aria (BD). 3x10 5 Dog CD45RB high T cells and 1x10 5 Dog T reg Cells were injected intravenously. Mice were administered 350 μg of α-CTLA-4 or isotype control or 600 μg of CTLA-4 nAb or ISVD control in ip twice a week. Mice were monitored and body weight was measured for 7 weeks after injection.

[0349] Intestinal permeability

[0350] Mice were tube-fed FITC-dextran (4 kDa, Sigma-Aldrich) 4 hours prior to the fluorescence measurement of FITC in serum.

[0351] Isolation of colon lamina propria, skin, and tumor cells

[0352] First, epithelial cells were removed by incubating 0.5-cm intestinal tissue fragments in a Hank buffered salt solution containing 5 mM EDTA and 10 mM HEPES at 37°C for 20 minutes, and then colonic lamina propria cells were isolated by repeating this incubation once more. The remaining tissue was cut into small fragments and digested under the same conditions in HBSS 1X medium containing 0.250 mg / mL Riverase (Roche), 30 U / mL DNase I (Sigma-Aldrich), and Dispase (Corning). The resulting cell suspension was stacked under a 40% / 80% percol gradient and centrifuged at 600 g for 10 minutes; LP cells were recovered from the interface.

[0353] Ear skin was shredded and digested in HBSS 1X medium containing 0.250 mg / mL Riverase (Roche), 30 U / mL DNase I (Sigma-Aldrich), and Dispase (Corning) at 37°C for 90 minutes. The cell suspension was filtered and washed twice with HBSS 1X buffer. The tumor was shredded and digested in HBSS 1X medium containing 0.250 mg / mL Riverase (Roche), 30 U / mL DNase I (Sigma-Aldrich), and Dispase (Corning) at 37°C for 30 minutes. The cell suspension was filtered and washed twice with HBSS 1X buffer.

[0354] Tissue structures from 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 to 4–5 μm, and stained with hematoxylin and eosin (H&E). The colon was scored for disease severity by a pathologist in a blind manner according to three criteria: Inflammation: When present, characterized by a large number (60–70%) of mononuclear cells (macrophages and lymphocytes) and 30–40% infiltration of neutrophils and band cells. Scoring of inflammation included the severity of infiltration, loss of glands, erosion, dilation of the glandular lumen, presence of pituitary abscess, and regression of epithelial cells. Inflammation was scored on a scale of 0–4, 0 = negative; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. Apoptosis: The prevalence of apoptotic bodies was scored on a scale of 0-3, 0 = negative; 1 = low; 2 = moderate; 3 = high. Regeneration: Evaluated regeneration changes included scoring the prevalence of mitotic phases in the upper 1 / 3 of the mucosa, nuclear density within individual glandular structures (nuclear density), and regularity of the surface epithelium. Apoptosis was scored on a scale of 0-3, 0 = negative; 1 = low; 2 = moderate; 3 = high.

[0355] Flow cytometry and antibodies

[0356] Cells were resuspended in PBS and stained with fixable viability stain (BD Bioscience) on ice in a dark room for 30 minutes. Then, cells were resuspended in staining buffer (FBS, BD Bioscience) and stained with various combinations of direct fluorescent dye-conjugation on ice for 30 minutes. For intracellular antigens, surface-stained cells were translucent, fixed on ice with Foxp3 staining buffer set (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 E-Biosciences. For all samples, acquisition was performed on an LSR II flow cytometer (BD). The data was analyzed using FLOWJO software (Tree Star).

[0357] Cells were stimulated with 500 ng / mL ionomycin and 50 ng / mL PMA (Sigma-Aldrich) when cytokine production was measured by flow cytometry. After 1 hour, Brefeldin A (Vidi Biosciences) was added for an additional 2 hours before staining.

[0358] Mouse Allogeneic Mixed Leukocyte Reaction (MLR) Test

[0359] 2.105 mouse C57B6 / J (8-12 weeks old, female) splenic T cells were isolated using the EASYSEP mouse T cell isolation kit (STEMCELL), and 1 x 10⁶ cells were isolated in the presence of indicated concentrations of α-CTLA-4, α-CTLA-4 (D265A), CTLA-4 nAb, or isotype controls. 5 They were co-cultured with irradiated Balb / c mouse splenocytes (at 2000 rad). On day 3, the supernatant was collected, and IL-2 and IFN-gamma production were measured by ELISA according to the manufacturer's protocol (Meso Scale Discovery). Subsequently, the cells were [for 6 hours or 16-18 hours] 3 Pulsed with [H]-thymidine (1 μCi per well). Cells were harvested from a glass fiber filter using a cell harvester. The filters were counted on a MicroBeta plate counter (PerkinElmer MicroBeta 2450) according to the manufacturer's instructions.

[0360] Total RNA isolation from tissues and cells and subsequent gene expression analysis using the Fluidigm BIOMARK platform

[0361] For real-time PCR analysis, total RNA was isolated using one of two methods. After homogenizing organs in RNA STAT-60 (Tel-Test Inc., Friendswood, Texas, USA) using a Polytron homogenizer, RNA extraction was performed using the MagMAX-96 for microarray kit (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. For cell samples, RNA was isolated using the ARCTURUS PICOPURE RNA isolation kit (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions.

[0362] DNase-treated total RNA was reverse transcribed using QUANTITECT Reverse Transcription (Qiagen, Valencia, California, USA) according to the manufacturer's instructions. Primers were obtained commercially from Thermo Fisher Scientific (Foster City, California, USA). Gene-specific pre-amplification was performed on at least 2 ng of cDNA according to the manufacturer's instructions for the Fluidigm Biomark (Fluidigm, Foster City, USA). Subsequently, real-time quantitative PCR was performed on the Fluidigm Biomark using two unlabeled primers at 900 nM and 250 nM of FAM-labeling probes (Thermo Fisher Scientific, Foster City, California, USA) along with the TAQMAN Universal PCR Master Mix containing UNG. Samples and primers were run on 48x48 or 96x96 arrays according to the manufacturer's instructions (Fluidigm, Foster City, USA). Ubiquitin levels were measured in a separate reaction and used to normalize the data by the ΔCt method. (Using the mean cycle threshold value for ubiquitin and the gene of interest for each sample, Equation 1.8 ^ (Ct ubiquitin - Ct gene of interest) x 10 4 Normalized values ​​were obtained using ). Primer reference sequences are available upon request.

[0363] statistics

[0364] Two-tailed paired and independent sample t-tests were used to calculate statistical significance in the remainder of this study. * P<0.05, ** P<0.01, *** P<0.001. Statistics were performed using GraphPad Prism 7 software.

[0365] Example 2

[0366] The antitumor efficacy of CTLA-4 nAb was evaluated in a mouse synonymous MB49 tumor model. MB49 cells are a urothelial carcinoma cell line derived from adult C57BL / 6 mice by exposing primary bladder epithelial cell exfoliations to 7,12-dimethylbenz[a]anthracene (DMBA) for 24 hours followed by long-term culture. Synonymous murine models of bladder cancer have been widely used for over 35 years.

[0367] MB49 mouse bladder cancer cells were implanted subcutaneously (sc) into 80 mice, and the animals were assigned to five treatment groups, each containing 10 mice. When the median starting tumor volume reached 103 mm³, mice were administered sc injections once every 4 days for a total of 4 doses. An unrelated control ISVD (30 mg / kg, lot number 01AQL) and a 5 mg / kg mIgG1 isotype control mAb (lot number 64AIS) were administered as treatment controls. Treatment included 30 mg / kg CTLA-4 nAb, 10 mg / kg Fc-eligible α-CTLA-4 (D265A), 5 mg / kg α-PD-1, or a combination of a CTLA4 targeting agonist and α-PD-1. Tumor growth was monitored for 21 days after the initiation of treatment.

[0368] Figure 9a presents the individual animal tumor volumes for each treatment group. Complete response (CR) up to day 21 is presented for the responsive treatment group. Figure 9b presents the mean tumor volume and the standard error of the mean for each treatment group (number of starters n=10 / group). Tumor volumes of animal forms removed from the study due to large tumor volumes were carried over to the mean until the last measurement for the corresponding treatment group was performed. Figures 9a-9b suggest that combination therapy with Fc-free CTLA-4 nAb and α-PD-1 provided strong antitumor benefits independent of Fc function, as in the CT26 colon tumor model (see Figure 3a), MB49 bladder tumor model, and MC38 colon tumor model.

[0369] Example 3

[0370] The antitumor efficacy of CTLA-4 nAbs was evaluated in a mouse syngeneic MC38 tumor model. MC38 mouse colon cancer cells were subcutaneously transplanted into 80 mice, and the animals were assigned to five treatment groups, each containing 10 mice. When the median starting tumor volume reached 246 mm³, mice were subcutaneously injected with the treatment at doses administered once every 4 days for a total of 4 doses. An unrelated control ISVD (30 mg / kg) and a 5 mg / kg mIgG1 isotype control mAb were administered as treatment controls. Treatment included 30 mg / kg CTLA-4 nAb, 10 mg / kg Fc-eligible α-CTLA-4 (D265A), 5 mg / kg α-PD-1, or a combination of a CTLA4 targeting agonist and α-PD-1. Tumor growth was monitored for 23 days after the initiation of treatment.

[0371] Figure 10a presents the individual animal tumor volumes for each treatment group. Complete response (CR) up to day 23 is presented for the responsive treatment group. Figure 10b presents the mean tumor volume and the standard error of the mean for each treatment group (number of starters n=10 / group). Tumor volumes of animal forms removed from the study due to large tumor volumes were carried over to the mean until the last measurement for the corresponding treatment group was performed. Figures 10a-10b suggest that combination therapy with Fc-free CTLA-4 nAb and α-PD-1 provided strong antitumor benefits independent of Fc function, as in the CT26 colon tumor model (see Figure 3a), MB49 bladder tumor model, and MC38 colon tumor model.

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[0391] 참고문헌

[0392] Azuma et al., B70 antigen is a second ligand for CTLA-4 and CD28. Nature. 1993 Nov 4;366(6450):76-9.

[0393] Collins et al., The interaction properties of costimulatory molecules revisited. Immunity. 2002 Aug;17(2):201-10.

[0394] Hui et al., T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017 Mar 31;355(6332):1428-1433.

[0395] Ingram et al., Anti-CTLA-4 therapy requires an Fc domain for efficacy. Proc Natl Acad Sci U S A. 2018 Apr 10;115(15):3912-3917.

[0396] Kamphorst et al., Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017 Mar 31;355(6332):1423-1427.

[0397] Koenitzer et al., Reformatting Rituximab into Human IgG2 and IgG4 Isotypes Dramatically Improves Apoptosis Induction In Vitro. PLoS One. 2015 Dec 29;10(12):e0145633.

[0398] Lanier et al., CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J Immunol. 1995 Jan 1;154(1):97-105.

[0399] Leach et al., Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996 Mar 22;271(5256):1734-6.

[0400] Pai et al., Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-relatedtoxicity. J Clin Invest. 2019 Jan 2;129(1):349-363.

[0401] Ribas &Wolchok, Cancer immunotherapy using checkpoint blockade. Science. 2018 Mar 23;359(6382):1350-1355.

[0402] Schneider-Merck et al., Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage. J Immunol. 2010 Jan 1;184(1):512-20.

[0403] Selby et al., Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res. 2013 Jul;1(1):32-42.

[0404] Sharma et al., Anti-CTLA-4 Immunotherapy Does Not Deplete FOXP3+ Regulatory T Cells (Tregs) in Human Cancers. Clin Cancer Res. 2018 Jul 27.

[0405] Simpson et al., Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med. 2013 Aug 26;210(9):1695-710.

[0406] Ullman-Cullere & Foltz, Body condition scoring: a rapid and accurate method for assessing health status in mice. Lab Anim Sci. 1999 Jun;49(3):319-23.

[0407] Vargas et al., Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies. Cancer Cell. 2018 Apr 9;33(4):649-663.e4.

[0408] Waight et al., Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens. Cancer Cell. 2018 Jun 11;33(6):1033-1047.e5.

[0409] It should be understood that while the present invention is described herein with reference to the illustrated embodiments, the invention is not limited thereto. Those skilled in the art and those familiar with the teachings of this invention will recognize additional variations and embodiments within the scope of the invention. Accordingly, the invention is limited only by the claims appended herein.

Claims

Claim 1 A pharmaceutical composition comprising a PD1 blocker and a CTLA blocker for use in a combination therapy for treating cancer in subjects requiring cancer treatment, wherein the combination therapy does not cause moderate to severe cutaneous or intestinal inflammatory immune-related toxicity, wherein the PD1 blocker is an anti-PD-1 antibody comprising a heavy chain (HC) having the amino acid sequence presented in SEQ ID NO: 27 and a light chain (LC) having the amino acid sequence presented in SEQ ID NO: 28, or an HC having the amino acid sequence presented in SEQ ID NO: 25 and an LC having the amino acid sequence presented in SEQ ID NO: 26, and the CTLA blocker is (i) an HC having a variable domain (VH) having the amino acid sequence presented in SEQ ID NO: 7 and an HC having an invariant domain having the amino acid sequence presented in SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 116 or 117, and (ii) A pharmaceutical composition comprising an effector-silencing anti-CTLA-4 antibody comprising an LC variable domain (VL) having the amino acid sequence presented in sequence identification number: 8 and an LC having an LC constant domain having the amino acid sequence presented in sequence identification number:

57. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A pharmaceutical composition according to claim 1, wherein the CTLA blocker is administered at a dose of more than 1 mg per kg, or at a fixed dose of 100 mg or more that does not depend on the body weight of the individual. Claim 6 A pharmaceutical composition according to claim 1, wherein the PD1 blocker is administered at (i) a dose containing 2 or 3 mg per kg, or a fixed dose of 200 mg that does not depend on the body weight of the individual, (ii) a dose containing more than 3 mg per kg, or a fixed dose of more than 200 mg that does not depend on the body weight of the individual, or (iii) a fixed dose of 200 mg to 400 mg that does not depend on the body weight of the individual. Claim 7 A pharmaceutical composition according to claim 1, wherein a PD1 blocker is administered to an individual first and a CTLA blocker is administered to an individual second; a CTLA blocker is administered to an individual first and a PD1 blocker is administered to an individual second; or a PD1 blocker and a CTLA blocker are jointly administered to an individual. Claim 8 A pharmaceutical composition according to claim 1, wherein the individual additionally receives a chemotherapy agent before, after, or in combination with combination therapy. Claim 9 A pharmaceutical composition according to claim 1, wherein the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastric cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, renal cancer, Hodgkin 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. Claim 10 A pharmaceutical composition according to claim 1, wherein the cancer is pancreatic cancer, bronchial cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, cancer of the uterus or endometrium, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, cancer of the small intestine or appendix, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete

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