TW202019965A - Method of treating multiple myeloma - Google Patents

Abstract

The disclosure provides a method of treating multiple myeloma, the method comprising administering to a subject in need thereof a heterodimeric antibody that binds CD3 and CD38.

Description

Treatment of multiple myeloma

Multiple myeloma is a neoplastic plasma cell disease characterized by the colonization and proliferation of malignant plasma cells in the bone marrow (BM) microenvironment, single protein in blood or urine, and related organ dysfunction. Multiple myeloma accounts for 1%-2% of all new cancer diagnoses, and accounts for about 20% of the total deaths from hematological malignancies. This disease is slightly more common in men and African Americans. Although recent improvements in the understanding of the pathogenesis of myeloma have developed new treatments and improved survival rates, multiple myeloma remains an incurable cancer.

In addition to the evidence of 10% or more selection of plasma cells or plasma cell tumors confirmed by biopsy in the BM examination, the diagnosis of multiple myeloma also requires the presence of one or more myeloma-defined events (MDE). MDE includes so-called CRAB (hypercalcemia, renal failure, anemia or osteolytic lesions) characteristics and three specific biomarkers: colonized BM plasma cells ≥ 60%, serum free light chain (sFLC) ratio ≥ 100 (The premise is that the affected sFLC level is ≥ 100 mg/L). Magnetic resonance imaging (MRI) shows more than 1 lesion (Rajkumar et al., 2011). Some genetic abnormalities occurring in tumor plasma cells play a major role in the pathogenesis of myeloma and determine the prognosis of the disease (Palumbo and Anderson, 2011).

Uncontrolled growth of myeloma cells has many consequences, including bone destruction, BM failure, increased plasma volume and viscosity, inhibition of normal immunoglobulin production, and kidney damage (Durie, 2011).

Symptomatic (active) disease should be treated immediately, while asymptomatic (smoldering) myeloma only requires clinical observation, because there is no obvious benefit from early conventional chemotherapy. Currently, research trials are evaluating the ability of immunomodulatory drugs to delay asymptomatic progression to symptomatic myeloma. For active myeloma, the current data support the introduction of induction therapy regimens (including thalidomide, lenalidomide, and/or bortezomib), and then to those that can tolerate autologous hematopoietic stem cell transplantation (HSCT) adjustment regimens Patients undergo autologous HSCT after their major disease response. The consideration of the physiological age (which may be different from the actual age) and the presence of coexisting conditions drive the choice of treatment and drug dosage. For example, for patients with significant comorbidities (including cardiopulmonary or liver injury), less intensive methods are applied to limit treatment-related mortality and reduce the risk of treatment interruption.

The treatment of relapsed/refractory multiple myeloma presents special treatment challenges due to the heterogeneity of the disease at the time of relapse and the lack of clear biological-based recommendations for choosing salvage therapy at various points in the progression of the disease. As awareness of the inherent colonization heterogeneity and genomic instability of plasma cells that affect innate and acquired treatment resistance increases, the identification of optimal selection and treatment sequence becomes critical. In recent years, some new drugs (including proteasome inhibitors (Carfilzomib and Ixazomib)), thalidomide derivatives pomalidomide, and groups for relapsed/refractory myeloma The protein deacetylase inhibitor panobinostat has been approved by the US Food and Drug Administration (FDA), which adds to the complexity of the above decision. Other molecular targeted therapies targeting specific cell communication pathways, as well as survival and proliferation control agents (including PI3K/AKT/mTOR inhibitors, Hsp90 inhibitors, cyclin-dependent kinase inhibitors, kinesin spindle protein inhibition) are currently being developed Agent). Despite the progress made in the management of multiple myeloma, almost all patients inevitably relapse. Each relapse of myeloma is usually more aggressive, leading to the development of refractory diseases that are associated with shorter survival times (Dimopoulos et al., 2015). Therefore, additional treatment options are required.

The present disclosure provides a method of treating multiple myeloma in a subject in need, such as a subject with relapsed/refractory multiple myeloma. The method includes administering to the subject about 0.05 mg to about 200 mg of heterodimeric antibody, the heterodimeric antibody comprising a) a first monomer comprising a first Fc domain and an anti-CD3 scFv, the anti- CD3 scFv comprises (i) a scFv variable light chain domain comprising vlCDR1 as shown in SEQ ID NO: 15, vlCDR2 as shown in SEQ ID NO: 16, and vlCDR3 as shown in SEQ ID NO: 17, (ii) scFv variable light chain domain comprising vhCDR1 as shown in SEQ ID NO: 11, vhCDR2 as shown in SEQ ID NO: 12, and vhCDR3 as shown in SEQ ID NO: 13, wherein the structure is used A domain linker covalently attaches the scFv to the N-terminus of the Fc domain; b) a second monomer comprising i) an anti-CD38 heavy chain variable domain comprising VhCDR1 as shown in SEQ ID NO: 65, vhCDR2 as shown in SEQ ID NO: 66, and vhCDR3 as shown in SEQ ID NO: 67, and ii) a heavy chain constant domain containing a second Fc domain; And c) a light chain comprising a variable constant domain and an anti-CD38 variable light chain domain, the anti-CD38 variable light chain domain comprising vlCDR1 as shown in SEQ ID NO: 69, as shown in SEQ ID NO: 70 Shown vlCDR2, and vlCDR3 shown in SEQ ID NO: 71. In various aspects, the dosage ranges from about 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg, or 200 mg, or about 36 mg to about 200 mg.

As needed, the method includes administering two doses of heterodimeric antibody to the subject weekly during the first and second weeks of treatment, and weekly to the subject during the third and fourth weeks of treatment A dose of heterodimeric antibody is administered, and from week 5 to the end of treatment, a dose of heterodimeric antibody is administered every two weeks. In various aspects, the treatment period (ie, the time at which multiple doses of heterodimeric antibody are administered to the subject) includes about 6 months to about 12 months, such as about 8 months. In some aspects of the disclosure, the heterodimeric antibody is administered by intravenous infusion over about 30 minutes to about 4 hours. For example, as needed, the first dose of heterodimeric antibody is administered in about four hours, the second dose of heterodimeric antibody is administered in about two hours, and all subsequent doses are administered in about 30 minutes Time to give. In some aspects, wherein multiple doses (ie, two or more) of heterodimeric antibody are administered to the subject during treatment, the dose of heterodimeric antibody is increased at least once during the treatment.

In some aspects, the method further includes administering dexamethasone to the subject. Dexamethasone is administered intravenously or orally, for example. If administered intravenously, dexamethasone is administered to the subject as needed within one hour before the administration of the heterodimeric antibody. Suitable dexamethasone doses include but are not limited to about 8 mg or about 4 mg.

In various aspects, the subject has previously been treated with an anti-CD38 monospecific antibody, such as daratumumab. In this regard, the subject is administered anti-CD38 monospecific antibody and the initial dose of heterodimeric antibody is administered after the washout period, which is sufficient to reduce the systemic concentration of anti-CD38 monospecific antibody To 0.2 μg/ml or lower, although this is not required in all aspects of this disclosure. No anti-CD38 monospecific antibody was administered during the elution (ie, anti-CD38 monospecific antibody treatment ended, the elution was allowed for a period of time, and then the initial dose of heterodimeric antibody was administered). In some cases, the method includes stopping treatment with the anti-CD38 monospecific antibody for at least 12 weeks (eg, about 13-15 weeks) before administering the initial dose of heterodimeric antibody (ie, administering to the subject Anti-CD38 monospecific antibody, then the initial dose of heterodimeric antibody is administered at a time point of at least 12 weeks after stopping the administration of anti-CD38 monospecific antibody). In various aspects, subjects were previously treated with proteasome inhibitors and/or immunomodulatory drugs.

In various embodiments, the anti-CD3 scFv comprises a variable heavy chain domain comprising an amine that is at least 90% identical (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 10 Acid sequence. In various aspects, the anti-CD3 scFv includes a variable heavy chain domain that includes an amino acid sequence that is included compared to SEQ ID NO: 10, optionally outside the CDR The region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. If desired, the anti-CD3 scFv includes a variable light chain domain that includes an amino acid that is at least 90% identical (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 14 sequence. In various aspects, the anti-CD3 scFv comprises a variable light chain domain comprising an amino acid sequence which is included compared to SEQ ID NO: 14, optionally outside the CDR The region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. For example, in various embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 90% (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 18. In various aspects, the anti-CD3 scFv contains an amino acid sequence that includes 1,2,3,4,5,6,7 as compared to SEQ ID NO: 18, optionally outside the CDR , 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions. In some aspects of the disclosure, the scFv domain linker is a charged linker. If desired, the anti-CD38 variable light chain domain comprises an amino acid sequence that is at least 90% identical (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 68 and/or the anti-CD38 heavy chain variable The domain comprises an amino acid sequence that is at least 90% identical (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 64. For example, the anti-CD38 variable light chain domain comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, SEQ ID NO: 68 11, 12, 13, 14, or 15 amino acid substitutions, and/or the anti-CD38 heavy chain variable domain comprises an amino acid sequence, which contains 1, 2, compared to SEQ ID NO: 64 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid substitutions. If necessary, such substitutions are outside the CDR. For example, in various aspects, the first monomer comprises an amino acid sequence at least 90% (eg, 100% identical) to the amino acid sequence shown in SEQ ID NO: 335 and/or the second monomer comprises the amino acid sequence of SEQ ID NO : The amino acid sequence shown in 82 is at least 90% identical (e.g., 100% identical) to the amino acid sequence and/or light chain comprising at least 90% identical to the amino acid sequence shown in SEQ ID NO: 84 (e.g. , 100% identical) amino acid sequence. In some embodiments, the first Fc domain and the second Fc domain of the heterodimeric antibody comprise one or more mutations that reduce homodimerization. In some embodiments, the first Fc domain and the second Fc domain comprise a variant group selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q, where S364K/E357Q: L368D/K370S represent preferred embodiments. In other aspects, the first and second Fc domains comprise amino acid substitutions E233P/L234V/L235A/G236del/S267K. If necessary, the heavy chain constant domain contains amino acid substitutions N208D/Q295E/N384D/Q418E/N421D.

Cross-reference and incorporation of related applications

This application claims priority from US Provisional Patent Application No. 62/698,675 filed on July 16, 2018, the contents of which are incorporated herein by reference.

This application is incorporated by reference into the International Patent Publication No. WO 2016/086196 filed on November 25, 2015; US Patent Publication No. 20160215063 filed on November 25, 2015; International Patent filed November 23, 2016 Publication No. WO 2017/091656; and US Patent No. 9,822,186 filed on March 30, 2015, the entire contents of which are expressly incorporated herein by reference, with particular reference to the drawings, legends, and patent application scope therein.

Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted at the same time as this article. Its identification is as follows: an ASCII (text) file named "52589_Seqlisting.txt", 707,436 bytes, created On July 15, 2019.

The present disclosure provides a method of treating multiple myeloma in a subject in need. This method uses a heterodimeric antibody that co-joins CD3 and CD38 in such a way as to temporarily connect malignant cells with T cells, thereby inducing T cells to mediatedly kill the bound malignant cells. The CD3 targeting method shows considerable hope, but the common side effect of this therapy is the related production of cytokines, which usually leads to toxic cytokine release syndrome. Because the anti-CD3 binding domain of the bispecific antibody engages all T cells, a subpopulation of CD4 T cells that produce high interleukins is recruited. In addition, CD4 T cell subsets include regulatory T cells, whose recruitment and expansion can potentially lead to immunosuppression and have a negative impact on long-term tumor suppression. One possible way to reduce interleukin production and possibly reduce CD4 T cell activation is to reduce the affinity of the anti-CD3 domain for CD3. However, too much reduced affinity may result in reduced efficacy of anti-CD3 domain containing therapeutic agents. The affinity of the bispecific antibody for CD38 also has an effect on the efficacy of the antibody in targeting cells expressing CD38. The methods described herein utilize heterodimeric antibodies that bind CD3 and CD38 in such a way as to maximize the destruction of target cells while reducing undesirable side effects (eg, uncontrolled release of interleukins).

The heterodimeric antibodies described herein use different monomers that contain amino acid substitutions, which make the formation of heterodimers "skew" compared to homodimers, As outlined more fully below, plus "pI variants" (which allow simple purification of heterodimers and separation of homodimers). Heterodimeric antibodies optionally contain engineered Fc domains or variant Fc domains, which self-assemble in production cells to produce heterodimeric proteins.

Various aspects of this method are described below. The use of section headings is for readability only, not to limit itself. The entire document is intended to be regarded as a unified disclosure, and it should be understood that all combinations of features described herein can be considered. Heterodimeric antibody

The method includes administering a heterodimeric antibody to a subject in need, the heterodimeric antibody comprising a) a first monomer comprising a first Fc domain and an anti-CD3 scFv. scFv contains variable heavy chain, scFv linker and variable light chain domain. If necessary, the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, which is attached to the N-terminus of the variable heavy chain (N-vh-linker-vl-C), although Can change the configuration (N-vl-linker-vh-C). Therefore, the description and description of scFv specifically include scFv in any orientation. In various aspects, the scFv domain linker is a charged linker. Many suitable scFv linkers can be used, and many are listed in the figures. Charged scFv linkers can be used to promote pI separation between the first and second monomers. That is, by incorporating charged scFv linkers, positive or negative (or both in the case of using scFv scaffolds for different monomers), this allows monomers containing charged linkers to change pI while No further changes to the Fc domain.

A domain linker was used to covalently attach the scFv to the N-terminus of the Fc domain. The "domain linker" connects any two domains outlined in this article. If desired, charged domain linkers can be used. Charged domain linkers can also, for example, increase the pi separation of the disclosed monomers, and therefore those included in the drawings can be used in any of the embodiments that utilize linkers herein.

The linker peptide may mainly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length suitable for connecting two molecules so that they assume the correct conformation with respect to each other so that the two molecules maintain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, it was found that a linker with a length of 1 to 20 amino acids can be used, and in some embodiments from about 5 to about 10 amino acids. Useful linkers include glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 332), (GGGGS)n (SEQ ID NO: 333), and (GGGS) n (SEQ ID NO: 334), where n is at least one (usually an integer from 3 to 4), glycine-alanine polymer, alanine-serine polymer and other flexible linkers. Alternatively, various non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol can be used as linkers.

Other linker sequences may include any sequence of any length of the CL/CH1 domain (but not including all residues of the CL/CH1 domain); for example, the first 5-12 amino acid residues of the CL/CH1 domain . The linker may be derived from an immunoglobulin light chain, such as Cκ or Cλ. The linker can be derived from any isotype of immunoglobulin heavy chain, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (eg, TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.

The anti-CD3 scFv comprises (i) a scFv variable light chain domain comprising vlCDR1 as shown in SEQ ID NO: 15, vlCDR2 as shown in SEQ ID NO: 16, and vlCDR3 as shown in SEQ ID NO: 17, And (ii) an scFv variable heavy chain domain comprising vhCDR1 shown in SEQ ID NO: 11, vhCDR2 shown in SEQ ID NO: 12, and vhCDR3 shown in SEQ ID NO: 13. If desired, the anti-CD3 scFv contains a variable heavy chain domain that contains at least 90% of the amino acid sequence shown in SEQ ID NO: 10 (eg, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) amino acid sequence. In various aspects, the anti-CD3 scFv includes a variable heavy chain domain that includes an amino acid sequence that is included compared to SEQ ID NO: 10, optionally outside the CDR The region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. If desired, the anti-CD3 scFv contains a variable light chain domain that contains at least 90% of the amino acid sequence shown in SEQ ID NO: 14 (eg, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) amino acid sequence. In various aspects, the anti-CD3 scFv comprises a variable light chain domain comprising an amino acid sequence which is included compared to SEQ ID NO: 14, optionally outside the CDR The region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In this regard, in various embodiments, the anti-CD3 scFv comprises the variable heavy chain domain of SEQ ID NO: 10 and the variable light chain domain of SEQ ID NO: 14. If necessary, the variable heavy and variable light chain domains are connected by a scFv domain linker comprising the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 158). In this regard, in various embodiments, the anti-CD3 scFv comprises at least 90% identical to the amino acid sequence shown in SEQ ID NO: 18 (eg, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identical) amino acid sequence. In various aspects, the anti-CD3 scFv comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 compared to SEQ ID NO: 18 , 13, 14 or 15 amino acid substitutions. In various aspects, sequence variation that results in a percent identity with the reference sequence of less than 100% indicates a modification outside the CDR sequence. In various aspects, the scFv contains the sequences shown herein that belong to anti-CD3_H1.32_L1.47.

"Fc" or "Fc region" or "Fc domain" refers to a polypeptide that includes a constant region of an antibody that does not include an immunoglobulin domain of the first constant region and is part of a hinge in some cases. Therefore, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexibility at the N-terminus of these domains Hinge. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain contains the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). The heterodimeric antibody is preferably an IgG antibody (which includes several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 at its carboxy terminus, where it is numbered according to the European index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγR receptors or FcRn receptors.

In various aspects, the first monomer (ie, the first Fc domain and the anti-CD3 scFv) comprises at least 90% identical amino acid sequence as shown in SEQ ID NO: 335 (eg, with SEQ ID NO: 335 The amino acid sequences shown in 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are the same). In various aspects, sequence variation that results in a percent identity with the reference sequence of less than 100% indicates a modification outside the CDR sequence.

The heterodimeric antibody of this method further comprises b) a second monomer comprising i) an anti-CD38 heavy chain variable domain and ii) a heavy constant domain comprising a second Fc domain. The anti-CD38 heavy chain variable domain comprises the following CDR sequences: variable heavy chain (vh) CDR as shown in SEQ ID NO: 65, vhCDR2 as shown in SEQ ID NO: 66, and as shown in SEQ ID NO: 67 VhCDR3 shown. If necessary, the anti-CD38 heavy chain variable domain contains at least 90% of the amino acid sequence shown in SEQ ID NO: 64 (for example, 91% of the amino acid sequence shown in SEQ ID NO: 64, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) amino acid sequence. In various aspects, the anti-CD38 variable heavy chain domain comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, compared to SEQ ID NO: 64 10, 11, 12, 13, 14 or 15 amino acid substitutions. In various aspects, the second monomer (ie, the anti-CD38 variable heavy chain domain and the heavy constant domain comprising the second Fc domain) comprises at least 90 amino acid sequences as shown in SEQ ID NO: 82 % Identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 82 )'S amino acid sequence. In various aspects, sequence variation that results in a percent identity with the reference sequence of less than 100% indicates a modification outside the CDR sequence.

The heterodimeric antibody also contains c) a light chain that contains a variable constant domain and an anti-CD38 variable light chain (vl) domain. The anti-CD38 variable light chain domain comprises the following CDRs: vlCDR1 as shown in SEQ ID NO: 69, vlCDR2 as shown in SEQ ID NO: 70, and vlCDR3 as shown in SEQ ID NO: 71. If desired, the anti-CD38 variable light chain domain contains at least 90% of the amino acid sequence shown in SEQ ID NO: 68 (for example, 91% of the amino acid sequence shown in SEQ ID NO: 68, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) amino acid sequence. In various aspects, the anti-CD38 variable light chain domain comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, compared to SEQ ID NO: 68 10, 11, 12, 13, 14 or 15 amino acid substitutions. In some embodiments, the light chain (comprising a variable constant domain and an anti-CD38 variable light chain domain) comprises at least 90% identical amino acid sequence as shown in SEQ ID NO: 84 (eg, with SEQ ID NO: The amino acid sequence shown in 84: 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are the same). In various aspects, sequence variation that results in a percent identity with the reference sequence of less than 100% indicates a modification outside the CDR sequence.

In a preferred embodiment, the heterodimeric antibody comprises a first monomer containing an anti-CD3 scFv, the anti-CD3 scFv comprising an anti-CD3 variable light chain domain containing the amino acid sequence of SEQ ID NO: 14 and An anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 10; a second monomer comprising an anti-CD38 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 64, and The light chain of the variable light chain domain containing the amino acid sequence of SEQ ID NO: 68. For example, in one embodiment, the heterodimeric antibody comprises a first monomer containing the amino acid sequence of SEQ ID NO: 335, a second monomer containing the amino acid sequence of SEQ ID NO: 82, and containing The light chain of the amino acid sequence of SEQ ID NO: 84.

The heterodimeric antibody adopts a structure called "opener" in FIG. 1A. One heavy chain of the "opener" type contains scFv, and the other heavy chain is a "regular" Fab type, which contains variable heavy and light chains. The two chains are joined together by the use of amino acid variants in a constant region (eg, Fc domain, CH1 domain, and/or hinge region) that promotes the formation of heterodimeric antibodies. The "opener" format has several obvious advantages. Antibody analogs that depend on two scFv constructs often have stability and aggregation problems, which are alleviated in the present disclosure by adding "regular" heavy and light chain pairs. In addition, in contrast to the version that relies on two heavy chains and two light chains, incorrect pairing of heavy chains and light chains (eg, heavy 1 and light 2 pairing, etc.) does not cause problems.

In various aspects, heterodimeric antibodies include modifications that promote heterodimeric antibody formation (ie, reduce homodimerization), modulate antibody function, etc. compared to wild-type antibody domain sequences. Modifications are usually concentrated in the Fc domain (although this is not required). The modification is mentioned by the amino acid position of the substitution, deletion or insertion relative to the natural sequence. For example, N434S or 434S is substituted with the Fc domain of serine at position 434 relative to the parent Fc polypeptide, which is numbered according to the EU index. Similarly, M428L/N434S defines Fc modifications that replace M428L and N434S relative to the parental Fc polypeptide. The identity of the wild-type amino acid may be unspecified, in which case the aforementioned variant is called 428L/434S. The order of replacement provided is arbitrary, that is, for example, 428L/434S is the same as M428L/N434S, and so on. For all positions in relation to the antibody in question, unless otherwise indicated, the amino acid position numbering is according to the EU index. The EU index or the EU index as in the Kabat or EU numbering scheme refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA [Journal of the American Academy of Sciences] 63: 78-85, the entirety of which is cited by reference Incorporated into this article). Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring amino acids, and in some cases, can include synthetic amino acids. Examples include US Patent No. 6,586,207; International Patent Publication Nos. WO 98/48032; WO 03/073238; US 2004-0214988A1; WO 05/35727A2; WO 05/74524A2; JW Chin et al., (2002), Journal of the American Chemical Society [Journal of the American Chemical Society] 124: 9026-9027; JW Chin, and PG Schultz, (2002), ChemBioChem [Chemical Biochemistry] 11: 1135-1137; JW Chin, et al., (2002), PICAS United States of America [PICAS United States of America] 99: 11020-11024; and L. Wang, and PG Schultz, (2002), Chem. [Chemistry] 1-10, all incorporated by reference.

There are many mechanisms that can be used to produce heterodimeric proteins. The amino acid variants that cause heterodimers are called "heterodimerization variants." Heterodimerization variants may include spatial variants (for example, the "knobs and holes" variants described below or "skew" variants and the "charge pair" variants described below) as well as the "pI variants" "These variants allow the separation and purification of homodimers and heterodimers. As described generally in International Patent Publication No. WO 2014/145806 (the disclosure is incorporated herein by reference in its entirety and specifically used to discuss "heterodimerization variants"), useful mechanisms for heterodimerization include As described in WO 2014/145806, the "peel and mortar" ("KIH"; sometimes referred to herein as the "skew" variant), "electrostatic steering" or "charge pair", the pI variation described in WO 2014/145806 , And additional conventional Fc variants outlined in WO 2014/145806 and herein.

There are several basic mechanisms that can lead to the simplification of the purification of heterodimeric antibodies; one relies on the use of pI variants so that each monomer has a different pI, thus allowing isoelectric purification of AA, AB, and BB dimer proteins . Alternatively, some bracket styles, such as the "bottle opener" style, also allow for separation based on size. It is also possible to "bias" the formation of heterodimers relative to homodimers. Therefore, a combination of steric heterodimerization variants and pi or charge pair variants was found to be particularly useful in the present invention. pI (isoelectric point) variant

For pI variants, amino acid modifications can be introduced into one or both of the monomeric polypeptides; that is, the pI of one of the monomers (referred to herein as "monomer A") can be engineered away from monomer B, or single Both bodies A and B can be changed, the pI of monomer A increases and the pI of monomer B decreases. Either by removing or adding charged residues (for example, neutral amino acids are replaced by positively or negatively charged amino acid residues, for example, glycine is replaced by glutamate) or Changes in the pI of the two monomers, changing the charged residue from positive or negative to opposite charge (aspartic acid to ionic acid) or changing the charged residue to a neutral residue (for example, charge The loss of lysine to serine). Many such variations are shown in the figure. Such modifications produce sufficient changes in pi in at least one monomer so that the heterodimer can be separated from the homodimer. As understood by those skilled in the art, this can be achieved by using a "wild type" heavy chain constant region and a variant region that has been engineered to increase or decrease its pI(wt A-+ B or wt A--B), or by increasing one area and reducing other areas (A+ -B- or A- B+).

Thus, in various aspects, a heterodimeric antibody includes one or more modifications in one or more constant regions to incorporate an amino acid substitution ("pI variant" or "pI substitution") into one or Of the two monomers, the isoelectric point (pI) of at least one (if not both) monomer of the heterodimeric protein is changed to form a "pI antibody." If the pi of the two monomers differ by as little as 0.1 pH units, then 0.2, 0.3, 0.4, and 0.5 or greater are suitable, and separation of the heterodimer from the two homodimers can be achieved.

To achieve good separation, the number of pI variants included on each or both monomers depends in part on the starting pI of the component, for example, the starting pI of anti-CD3 scFv and anti-CD38 Fab. That is, in order to determine the monomer or "direction" to be engineered (eg, correction or negative), the Fv sequences of the two domains are calculated and a decision is made accordingly. Different Fvs will have different starting pis that can be utilized. In some embodiments, using the graph in FIG. 19 of US Patent Publication No. 2014/0370013, the change in pI is calculated based on the variable weight chain constant domain. Alternatively, the pi of each monomer can be compared. Generally, the pI is engineered to result in a total pI difference for each monomer of at least about 0.1 log, preferably 0.2 to 0.5.

A preferred combination of pI variants is shown in FIG. These changes are shown relative to IgG1, but all isotypes and isotype hybrids can be changed in this way. In the case where the heavy chain constant domain is derived from IgG2-4, R133E and R133Q can also be used.

In one embodiment, the Fab monomer (negative side) contains substitutions 208D/295E/384D/418E/421D (N208D/Q295E/N384D/Q418E/N421D (when relative to human IgG1)) and the scFv monomer (positive side) ) Contains positively charged scFv linkers, including (GKPGS) ₄ .

Modifications that regulate pI can also be made in the light chain. Amino acid substitutions for reducing the light chain pI include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E, and the addition of DEDE peptides at the C-terminus of the light chain. Changes in this category based on constant lambda light chains include one or more substitutions at R108Q, Q124E, K126Q, N138D, K145T, and Q199E. In addition, the pI of the light chain can also be increased. Skew/spatial variant

There are many suitable heterodimerization skew variant pairs. These variants appear in the form of "groups" and "pairs." That is, one group of the pair is incorporated into the first monomer, and the other group of the pair is incorporated into the second monomer. It should be noted that these groups do not necessarily appear to be "pestle and mortar" variants (there is a one-to-one correspondence between the residue on one monomer and the residue on the other monomer); An interface is formed between the two monomers, which promotes the formation of heterodimers and hinders the formation of homodimers, so that the percentage of spontaneously formed heterodimers under biological conditions exceeds 90% instead of the expected 50% (25% homodimer A/A: 50% heterodimer A/B: 25% homodimer B/B).

In some embodiments, the formation of heterodimers is promoted by adding spatial variants. That is, by changing the amino acid in each heavy chain, different heavy chains are more likely to combine to form a heterodimer structure rather than forming a homodimer with the same Fc amino acid sequence. Suitable examples of spatial variants are included in FIG. 9.

A mechanism commonly referred to in the art as "pestle and mortar" can also be used as needed, which refers to the engineering of amino acids that produce steric effects to promote heterodimer formation and are not conducive to homodimer formation. This is further described in US Patent Publication No. 20130205756, Ridgway et al., Protein Engineering [protein engineering] 9(7): 617 (1996); Atwell et al., J. Mol. Biol. [Journal of Molecular Biology] 1997 270: 26; US Patent No. 8,216,805, which is incorporated by reference in its entirety. The figure identifies many "monomer A-monomer B" pairs that depend on the "mortar". In addition, as described in Merchant et al., Nature Biotech. [Natural Biotech] 16: 677 (1998), these "pestle and mortar" mutations can be combined with disulfide bonds to form biased heterodimerization.

It was discovered that another mechanism for generating heterodimers is sometimes referred to as "electrostatic turning", as described in Gunasekaran et al., J. Biol. Chem. [Journal of Biochemistry] 285(25): 19637 (2010), The entirety is incorporated by reference. This is sometimes referred to as a "charge pair" in this article. In this embodiment, static electricity is used to bias the formation of heterodimerization. As those skilled in the art will understand, these may also have an effect on pI, and therefore purification, and therefore pI variants may also be considered in some cases. However, since these are produced to force heterodimerization and are not used as purification tools, they are classified as "spatial variants". These include, but are not limited to, D221E/P228E/L368E, paired with D221R/P228R/K409R (ie, the corresponding group of these monomers); and C220E/P228E/368E, paired with C220R/E224R/P228R/K409R.

The additional monomer A and monomer B variants can be combined with other variants as desired and independently, such as the pI variants outlined herein or other spaces shown in FIG. 37 of US Patent Publication No. 2012/0149876 Variants, the figure and the legend and its SEQ ID NO are expressly incorporated herein by reference.

In some embodiments, the spatial variants outlined herein can be incorporated into one or two monomers together with any pI variant (or other variants, such as Fc variants, FcRn variants, etc.) as needed and independently, And can be included in or excluded from the protein of the present invention independently and as necessary.

A list of suitable skewed variants can be found in Figures 9 and 12. Particularly useful in many embodiments are the following pairs, including but not limited to, S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q. In terms of naming, the "S364K/E357Q: L368D/K370S" pair means that one of the monomers has the double variant group S364K/E357Q and the other monomer has the double variant group L368D/K370S. Additional Fc variants for function adjustment

There are many useful Fc amino acid modifications that can be prepared for various reasons, including, but not limited to, changing the binding to one or more FcγR receptors, changing the binding to FcRn receptors, and so on.

Many useful Fc substitutions can be made to alter the binding to one or more FcyR receptors. Substitutions that lead to increased binding as well as decreased binding may be useful. For example, it is known that increased binding to FcγRIIIa generally leads to increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated response in which non-specific cytotoxic cells that exhibit FcγR recognize the binding antibody on the target cell and subsequently cause Lysis of target cells). Similarly, in some cases, reduced binding to FcyRIIb (inhibitory receptor) is also beneficial. Amino acid substitutions found useful in the present invention include those listed in US Patent Publication Nos. 2006/0024298 (especially FIG. 41), 2006/0121032, 2006/0235208, 2007/0148170, all of which are cited by reference Incorporated expressly in this article, and in particular the variants disclosed therein. Specific variants that can be used include, but are not limited to 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, additional Fc substitutions were found, which can be used to increase binding to FcRn receptors and increase serum half-life, as specifically disclosed in US Patent Publication No. 2009/0163699, which is incorporated herein by reference in its entirety, Such substitutions include but are not limited to 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.

Another class of functional variants are "FcγR ablation variants" or "Fc knockout (FcKO or KO)" variants. For some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all Fcγ receptors (eg, FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. That is, for example, particularly when using bivalent antibodies that monovalently bind to CD3, it may be desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. Consider any level of reduction (eg, 50%, 60%, 70%, 80%, 90%, or 100% reduction in binding or activity). Examples of ablation variant modifications are depicted in FIG. 11 and each can be included or excluded independently and as needed. The preferred aspect utilizes ablation variants selected from the group consisting of the following: G236R/ L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V L235A/G236del. It should be noted that the ablation variants cited herein eliminate FcγR binding, but generally do not eliminate FcRn binding. Additional antibody considerations

This disclosure considers the use of other heterodimeric antibodies in this method. For example, variable heavy and light sequences, as well as the scFv sequence described above (and Fab sequences containing such variable heavy and light sequences) can be used in other forms, such as in Figure 2 of International Patent Publication No. 2014/145806 Those depicted (the figures, formats and legends of which are expressly incorporated herein by reference), together with FIGS. 1A and 1B. In addition, the amino acid sequences of the CD3 binding region and the CD38 binding region (for example, CDR sequences, variable light chain and variable heavy chain sequences, and/or full-length heavy chain and light chain sequences) are provided in the sequence listing provided together Provided and summarized in Figure 22. This article considers any combination of the sequences cited in Figure 22, as long as the resulting heterodimeric antibody is conjugated to both CD3 and CD38. Anti-CD3/anti-CD38 antibodies are further described with reference to International Patent Publication No. WO 2016/086196; U.S. Patent Publication No. 20160215063; International Patent Publication No. WO 2017/091656; and U.S. Patent No. 9,822,186 are described, the reference is borrowed in its entirety Incorporated herein by reference, and in particular regarding the description of anti-CD3/anti-CD38 antibodies and their amino acid and nucleic acid sequences, sequence listings and figures.

With regard to CD3 binding, the heterodimeric antibody may comprise an anti-CD3 antigen binding domain with medium or "moderate" affinity for CD3 (which also binds CD38). In this regard, the affinity (KD) of the heterodimeric antibody to CD3 is about 15-50 nM (eg, about 16-50 nM, 15-45 nM, about 20-40 nM, about 25-40 nM, or Approximately 30-40 nM), if necessary, the affinity is measured using the assay described in US Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, which publication is incorporated herein by reference.

In another aspect, the heterodimeric antibody of the method comprises an anti-CD3 antigen binding domain, which is a "strong" or "high affinity" binding agent for CD3 (for example, an example is depicted as H1.30_L1.47 (see The heavy chain and light chain variable domains need to include appropriate charged linkers) and also bind CD38. In various embodiments, the antibody construct binds CD3 with an affinity (KD) of about 3-15 nM (eg, 3-10 nM or 4-7 nM), using US Patent Publication No. 20160215063 and international patents as needed The assay described in Publication No. WO 2017/091656 measures affinity, which publication is incorporated herein by reference. In other embodiments, the method uses a heterodimeric antibody comprising an anti-CD3 antigen binding domain that is a "light" or "low affinity" binding agent for CD3. In this regard, the affinity of the heterodimeric antibody to CD3 (KD) is about 51 nM or more (for example, about 51-100 nM) as needed, and US Patent Publication No. 20160215063 and International Patent Publication are used as needed The assay described in case number WO 2017/091656 measures affinity, and the disclosure is incorporated herein by reference.

The affinity of the bispecific antibody for CD38 also has an effect on the efficacy of the antibody in targeting cells expressing CD38. Bispecific antibodies with "moderate" or "low" affinity for CD38 can effectively kill target cells in vitro and in vivo, while having a reduced toxicity profile. In various embodiments, bispecific antibodies that demonstrate "high" affinity for CD38 bind to CD38 with an affinity (KD) of, for example, less than 1 nM; bispecific antibodies that demonstrate "moderate" or "medium" affinity for CD38 For example, an affinity (KD) of about 1-10 nM (eg, 2-8 nM or 3-7 nM) binds CD38; bispecific antibodies that demonstrate "low" or "light" affinity for CD38, for example, about 11 nM or more (Eg 11-100 nM) affinity (KD) combined with CD38, the full affinity is measured using the method shown in US Patent Publication No. 20160215063 and International Patent Publication No. WO 2017/091656, which is incorporated by reference and Into this article.

Generally, specific binding can be exhibited as, for example, an antibody has at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, A KD of at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M or higher, where KD refers to the rate of dissociation of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more than the control molecule relative to the antigen. Similarly, the specific binding of a specific antigen can be exhibited as, for example, relative to a control, the antibody has the following antigen or epitope KA or Ka for the antigen: at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000 times or more, where KA or Ka refers to the dissociation rate of a specific antibody-antigen interaction.

If desired, the heterodimeric antibody contains cysteine substituted serine at position 220; usually, this is on the "scFv monomer" side of the heterodimeric antibody, although it can also be on the "Fab monomer" Side or both to reduce disulfide formation. The sequences herein specifically include one or both of these substituted cysteines (C220S). Snippet

The present disclosure also considers the use of antibody fragments (unlike the full-length antibodies that make up the natural biological form of the antibody, the antibody fragments include variable and constant regions, which usually include Fab and Fc domains and optionally additional antigen binding structures Domain, such as scFv). Antibody fragments contain at least one constant domain, which can be engineered to produce heterodimers, such as pi engineering. Other antibody fragments that can be used include fragments containing one or more CH1, CH2, CH3, hinge, and CL domains of the invention that have been pi engineered. Chimerism/Humanization

The heterodimeric antibody may be a mixture from different species, such as chimeric antibodies and/or humanized antibodies. Generally, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, a "chimeric antibody" traditionally contains one or more variable regions from one (or in some cases, rats) and one or more constant regions from humans. "Humanized antibodies" generally refer to non-human antibodies that have variable domain framework regions that are exchanged with sequences found in human antibodies. Generally, in humanized antibodies, the entire antibody except the CDR is encoded by a polynucleotide of human origin or is the same as such antibody (except within its CDR). CDRs (partially or wholly encoded by nucleic acids derived from non-human organisms) are grafted into the β-sheet framework of the variable regions of human antibodies to produce antibodies whose specificity is determined by the grafted CDRs. The production of such antibodies is described in, for example, International Patent Publication No. WO 92/11018, Jones, 1986, Nature [Natural] 321: 522-525, and Verhoeyen et al., 1988, Science [Science] 239: 1534-1536. The disclosures are incorporated by reference in their entirety. It is usually necessary to "back-mutate" the selected acceptor framework residues to the corresponding donor residues in order to regain the lost affinity in the initially grafted construct (US Patent Nos. 5530101; 5585089; 5693861; 5693762; 6180370; 5859205; 5821337; 6054297; and 6407213, both of which are incorporated by reference in their entirety). Humanized antibodies also comprise at least a portion of the constant region of an immunoglobulin (typically human immunoglobulin), and therefore typically a human Fc region. Humanized antibodies can also be produced using mice with genetically engineered immune systems. Roque et al., 2004, Biotechnol. Prog. [Biotechnology Progress] 20: 639-654, incorporated by reference in its entirety. Various techniques and methods for humanizing and reconfiguring non-human antibodies are well known in the art (see Tsurushita and Vasquez, 2004, Humanization of Monoclonal Antibodies [Molecularization of Monoclonal Antibodies], Molecular Biology of B Cells [ Molecular Biology of B Cells], 533-545, Elsevier Science (Elsevier Science Press) (USA), and references cited therein, all of which are incorporated herein by reference). Humanization methods include but are not limited to those described in Jones et al., 1986, Nature [Natural] 321: 522-525; Riechmann et al., 1988; Nature [Natural] 332: 323-329; Verhoeyen et al., 1988, Science [ Science], 239: 1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA [Proceedings of the American Academy of Sciences] 86: 10029-33; He et al., 1998, J. Immunol. [Journal of Immunology] 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA [Proceedings of the American Academy of Sciences] 89: 4285-9, Presta et al., 1997, Cancer Res. [Cancer Research] 57(20): 4593-9; Gorman Et al., 1991, Proc. Natl. Acad. Sci. USA [Proceedings of the American Academy of Sciences] 88: 4181-4185; O’Connor et al., 1998, Protein Eng [Protein Engineering] 11: 321-8, all borrowed Incorporated by reference. Humanization or other methods to reduce the immunogenicity of variable regions of non-human antibodies may include surface resurfacing methods, as described, for example, in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA [Proceedings of the American Academy of Sciences] 91 : 969-973, incorporated by reference in its entirety. treatment solutions

Heterodimer antibodies are administered to subjects in need, such as human subjects with multiple myeloma, such as relapsed/refractory multiple myeloma. Recurrent myeloma is characterized by a relapse of the disease after a previous response. Examples of laboratory and radiological criteria for signaling diseases include but are not limited to serum or urine single protein (M protein) increase ≥ 25% or ≥ 25% from involved and uninvolved serum-free light chains from the lowest point Differences, or the development of new plasmacytomas or hypercalcemia. Sonneveld et al., Haematologica [Hematology]. April 2016; 101(4): 396-406. In patients with non-secretory diseases, the recurrence is characterized by an increase in bone marrow plasma cells. The signs of recurrent disease are also characterized by the appearance or recurrence of one or more CRAB criteria or rapid and consistent biochemical relapse. Refractory myeloma is a myeloma that does not respond to treatment. Relapsed/refractory multiple myeloma refers to a disease that becomes unresponsive or progresses at the time of treatment or within 60 days of the last treatment among patients who have previously produced at least a minimal response to previous therapy. Sonneveld, as mentioned above; Anderson et al., Leukemia. 2008; 22(2): 231-239.

The method of the present disclosure includes administering a dose of about 0.05 mg to about 200 mg of heterodimeric antibody to the subject. In various embodiments, the dosage is about 0.5 mg to about 200 mg, about 0.5 to about 150 mg, about 1 mg to about 150 mg, about 10 mg to about 100 mg, about 10 mg to about 200 mg, about 4 mg To about 200 mg, about 12 mg to about 200 mg, about 12 mg to about 100 mg, about 36 mg to about 200 mg, about 36 mg to about 100 mg, or about 100 mg to about 200 mg. In various aspects of the method, the dose administered to the subject is about 0.05 mg, about 0.15 mg, about 0.45 mg, about 1.35 mg, about 4 mg, about 12 mg, about 36 mg, about 100 mg, or about 200 mg.

In an alternative aspect, the single-dose heterodimeric antibody is at least about 0.05 mg, at least about 0.15 mg, at least about 0.45 mg, at least about 1.35 mg, at least about 4 mg, at least about 12 mg, at least about 36 mg, or At least about 100 mg. In various aspects, a single dose of heterodimeric antibody does not exceed about 200 mg (eg, does not exceed about 100 mg or does not exceed about 36 mg). It should be understood that a single dose can be administered by multiple administrations (ie, divided doses), such that the multiple administration combination is the dose described herein. For example, multiple administrations (eg, two or more injections) combine at least about 0.05 mg, at least about 0.15 mg, at least about 0.45 mg, at least about 1.35 mg, at least about 4 mg, at least about 12 mg, at least about 36 mg or at least about 100 mg. In various aspects, the multiple-dose combination of single-dose heterodimeric antibodies is no more than about 200 mg (eg, no more than about 100 mg or no more than about 36 mg).

In various aspects of the method, the dosage is adjusted during the treatment. For example, the subject administers the initial dose at one or more doses and uses a higher dose in one or more subsequent doses. In other words, the present disclosure considers increasing the dose of heterodimeric antibody at least once during the course of treatment. Alternatively, the dose can be reduced during the treatment so that as the treatment progresses, the amount of heterodimeric antibody decreases.

The present disclosure contemplates a method in which multiple (ie, two or more) doses of heterodimeric antibody are administered during treatment. The doses can be administered at any interval, for example, once a week, twice a week, three times a week, four times a week or five times a week. Individual doses can be administered every two weeks, every three weeks or every four weeks. In other words, in some aspects, a two-week waiting period elapses between administration of the heterodimeric antibody to the subject. The waiting period between dose administration need not be consistent during treatment. In other words, the interval between doses can be adjusted during treatment. In some aspects, the method includes administering two doses of heterodimeric antibody to the subject each week during the first and second weeks of treatment (ie, twice a week in Week 1 and Week 2), A dose of heterodimeric antibody is administered to the subject weekly during the third and fourth weeks of treatment (ie, once a week in weeks 3 and 4), and from week 5 to the end of treatment A dose of heterodimeric antibody is administered every two weeks (ie, from the 5th week to the end of treatment with a two-week waiting period between doses). Although not wishing to be bound by any particular theory, the shorter interval between the first dose administered (eg, two doses per week) promotes rapid target cell clearance. Increasing the dosage interval as described herein can maintain cell clearance while minimizing undesirable side effects associated with immunotherapy. Alternatively, in various aspects, the method includes administering a dose of heterodimeric antibody weekly during the first 1-4 weeks of treatment, and if necessary, administering a dose of heterodimeric antibody every two weeks from week 5 to the end of treatment Dimer antibody.

Multiple doses of heterodimeric antibody over, for example, three months to about 18 months, or about three months to about 12 months, or about three months to about nine months, or about three months to about six Months, or about three months to about eight months, or about six months to about 18 months, or about six months to about 12 months, or about eight months to about 12 months, or about six The treatment period is from about one month to about eight months, or from about eight months to about 12 months (eg, about eight months). If desired, multiple (ie, two or more) doses of heterodimeric antibody are administered over a treatment period of about 12 weeks to about 52 weeks, or about 12 weeks to about 36 weeks, or about 24 weeks to about 32 weeks , Where the dose is administered twice a week, once a week, once every two weeks or once every four weeks.

"Treatment" multiple myeloma means achieving any positive treatment response to the disease. For example, a positive treatment response includes one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) an inhibition of neoplastic cell survival; (4) an accessory tumor cell Reduced protein production; (5) Inhibition of tumor growth (ie, slowed to a certain extent, preferably stopped); (6) Increased patient survival; and (7) One or a disease or condition-related Various symptoms have been relieved. Can use methods such as magnetic resonance imaging (MRI) scans, x-ray imaging, computed tomography (CT) scans, bone scan imaging, endoscopy, and tumor biopsy sampling (including bone marrow aspiration (BMA) and circulating tumor cell count )'S screening technology to assess tumor response to changes in tumor morphology (ie, overall tumor burden, tumor size, etc.). A complete treatment response is not required (ie, there is no clinically detectable disease, any previous abnormal radiographic studies, normalization of bone marrow and cerebrospinal fluid (CSF), or abnormal individual protein); any degree of improvement is expected. Various other parameters related to disease treatment and improvement are listed in the examples.

Heterodimeric antibodies can be administered to the subject by any suitable means, for example, by intravenous, intraarterial, intralymphatic, intrathecal, intracerebral, intraperitoneal, intracerebrospinal, intradermal, subcutaneous, Intra-articular, intra-synovial, oral, topical or inhalation route. For example, the heterodimeric antibody can be administered as a bolus injection by intravenous administration or by continuous infusion for a period of time. In various aspects, the method includes administering the heterodimeric antibody by intravenous infusion for a period of about 30 minutes to about four hours. If necessary, reduce the infusion time during subsequent administration. For example, in one embodiment, the first dose of heterodimeric antibody is administered over a period of about four hours, and the subsequent dose is administered over a period of two hours or less. In this regard, the first dose of heterodimeric antibody is administered over a period of about four hours as needed, the second dose of heterodimeric antibody is administered over a period of about two hours as needed, and the subsequent dose is administered as required It will be administered after about 30 minutes.

In some cases, the subject has previously received treatment for multiple myeloma. For example, subjects may have previously been administered immunomodulatory drugs (thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (eg, pomalidomide, bortezomib, or carfilzomib) ), dexamethasone, doxorubicin or a combination thereof. If necessary, the subject was previously treated with an anti-CD38 monospecific antibody such as daratumumab (DARZALEX®). In various embodiments, the subject relapses or is refractory to previous anti-CD38 monospecific antibody treatment. When the patient has been treated with anti-CD38 monospecific antibody, it is preferable to administer the initial dose of heterodimeric antibody after the elution period, which is sufficient to reduce the systemic concentration of anti-CD38 monospecific antibody to 0.2 μg/ml or lower. In other words, the method includes a waiting period between previous administration of anti-CD38 monospecific antibody and administration of heterodimeric antibody. In various embodiments, the method includes stopping treatment with the anti-CD38 monospecific antibody for at least 12 weeks (eg, about 13 to about 15 weeks) prior to the administration of the initial dose of heterodimeric antibody. Composition

By combining antibodies with the required purity and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition [Remington Pharmaceutical Sciences 16th edition], Osol, A. Editor [1980] ) Mix to prepare a heterodimeric antibody formulation for storage in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffers (such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine Acid; preservatives (eg octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, bensonin chloride; phenol, butyl or benzyl alcohol; alkyl para-hydroxy Parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (Less than about 10 residues) polypeptide; protein, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, Asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannose Alcohol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg, Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^™ , PLURONICS ^™, or polyethylene dioxide Alcohol (PEG).

The formulation may also contain more than one active agent, preferably one or more active agents that do not adversely affect each other. For example, it may be necessary to provide antibodies with other specificities. Alternatively or additionally, the composition may contain cytotoxic agents, cytokines, growth inhibitors, and/or small molecule antagonists. Such molecules are suitably present in combination in amounts effective for the intended purpose. Combination therapy

As needed, part of a heterodimer anti-system treatment regimen, which includes administration of one or more other therapeutic agents, radiation therapy, stem cell transplantation, and the like.

The method of the present disclosure may further include administering dexamethasone to the subject as needed. Dexamethasone can be administered by any route, such as the route described herein. Preferably, dexamethasone is administered intravenously or orally. When dexamethasone is administered intravenously, the heterodimeric antibody is administered to the subject as needed within one hour before administration. Dexamethasone is administered in an amount of about 8 mg or about 4 mg as needed.

In various embodiments, the method of the present disclosure also includes administering a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (eg, irinotecan, topotecan, camptothecin and its analogs or metabolites, and many (Rubicin); topoisomerase II inhibitors (such as etoposide, teniposide, and daunorubicin); alkylating agents (eg, melphalan ), chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, Semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide) ; DNA intercalator (such as cisplatin, oxaliplatin (oxaliplatin) and carboplatin); DNA intercalator and free radical generator (such as bleomycin (bleomycin)); and nucleoside mimics (for example, 5- 5-fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, ), pentostatin and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide (lenalidomide) and related analogs (eg CC-5013 and CC-4047); tyrosine protein kinase inhibitors (eg imatinib mesylate and gefitinib); proteasome inhibitors (eg bortezomib (bortezomib), CEP-18770, MG132, peptide vinyl sock, peptide epoxy ketone (such as epoxomicin and carfilzomib), β-lactone inhibitor (such as lactosporin ( lactacystin), MLN 519, NPI-0052, Salinosporamide A), compounds that produce dithiocarbamate complexes with metals (such as disulfiram), and certain antioxidants (such as tableware) Catechin-3-gallate, catechin-3-gallate and Salinosporamide A); NF-κB inhibitors (including IκB kinase inhibitors); antibodies (eg, trastuzumab Trastuzumab, rituximab, cetuximab, and bevacizumab), which bind to overexpressed proteins in cancer, thereby downregulating cell replication; and known Other inhibitors of up-regulated, over-expressed or activated proteins or enzymes in cancer whose inhibition down-regulates cell replication.

The treatment regimen may include administration of other antibody therapeutics, such as erlotuzumab (humanized monoclonal antibody against SLAMF7; Tai et al., Blood, 2008; 112: 1329-37); CD38 targeting Daratumumab, MOR202, and isatuximab; nBT062-SMCC-DM1, nBT062-SPDB-DM4, and nBT062-SPP-DM1 targeting CD138; lucatol targeting CD40 Lucatumumab (also known as HCD122) and dacetuzumab (also known as SGN-40); Lorvotuzumab targeting CD56. For a review of antibody therapeutics for the treatment of multiple myeloma, see, for example, Tandon et al., Oncology & Hematology Review [2015, 11(2): 115-21 and Sondergeld et al. , Clinical Advances in Hematology & Oncology, 2015; 13(9), 599, both of which are incorporated by reference.

In some embodiments, the heterodimeric antibody is prior to treatment with Velcade® (bortezomib), Thalomid ^™ (thalidomide), Aredia ^™ (pamidronate), or Zometa ^™ (zoledronic acid) , At the same time or later.

All cited references are incorporated by reference in their entirety. Although specific embodiments of the present invention have been described above for illustrative purposes, those skilled in the art will understand that many changes can be made in details without departing from the invention described in the scope of the appended patent applications. Examples

This example is provided to further illustrate various aspects of the disclosed method. This example is not meant to limit the invention to any particular application or theory of operation. Preclinical studies

Heterodimeric antibody (hereinafter referred to as "Ab-A") (which includes a first monomer, a second monomer, and a light chain, the first monomer includes the amino acid sequence of SEQ ID NO: 335, the first The second monomer contains the amino acid sequence of SEQ ID NO: 82, and the light chain contains the amino acid sequence of SEQ ID NO: 84) is a highly specific and effective molecule that can induce human T cells to kill multiple CD38 positive For tumor cell lines, the half-maximum effective concentration (EC50) is in the range of 0.65 ± 0.3 ng/mL to 21.77 ± 5.22 ng/mL (5.2 pM to 174.2 pM). In the case of cynomolgus monkey T cells or cynomolgus monkey B cells against human cancer cells, the efficacy of Ab-A is in the range of 2.17 ± 0.4 ng/mL to 12.33 ± 0.68 ng/mL (17.6 pM to 98.46 pM) . The EC50 value for cytotoxicity against CD38 positive cells varies between tumor cell lines, and the difference between the most sensitive and least sensitive cell lines is 33.5 times. However, this comparison is based on measurements performed under different conditions, including different effector cells (peripheral blood mononuclear cells (PBMC) compared to purified T cells), different readings (based on flow compared to luciferase ) And cell lines from multiple indications (multiple myeloma, acute myeloid leukemia, B-cell lymphoma, histiocytic lymphoma). When the measurement conditions were standardized in four multiple myeloma cell lines with different CD38 surface expression levels, the difference between the most sensitive and the least sensitive multiple myeloma cell lines was much narrower, four-fold.

Ab-A effectively engages and activates human T cells in the presence of CD38 positive target cells, resulting in target lysis accompanied by increased expression of T cell markers CD69, CD25, and CD38, increased T cell size, T cell expansion, and proinflammatory Cytokines IFN-γ and TNF-α are released. Similar T cell activation was observed with cynomolgus monkey T cells or in autologous B cell depletion assays with cynomolgus monkey PBMC, and data from in vitro studies demonstrated the pharmacology of the cynomolgus monkey line for toxicity testing Related animal species. However, cynomolgus monkey T cells performed significantly higher than the CD38 level of human T cells. Although T cell expansion was observed in the human T cell redirected lysis assay, the number of T cells decreased or remained constant during a similar assay using cynomolgus monkey PBMC, indicating that Ab-A has no effect on T cells between two species Difference effect.

Soluble CD38 is present in two species at very different concentrations: in patients with multiple myeloma, the average sCD38 concentration is 0.39 ng/mL (compared to 0.085 ng/mL in healthy individuals), with a maximum detection value of 2.82 ng /mL (n = 44), while in cynomolgus monkeys, the serum sCD38 concentration ranged from 0 to 247.8 ng/mL. In vitro , Ab-A-induced redirected lysis is not affected by soluble human CD38 at a concentration of 2 ng/mL. The soluble cynomolgus monkey CD38 at a concentration of 200 ng/mL induced a moderately 2-fold increase in the redirected lysis EC50 of Ab-A. Therefore, sCD38 levels in patients with multiple myeloma are unlikely to interfere with Ab-A activity. In addition, in the absence of target cells in the presence of Ab-A, sCD38 at a concentration of 200 ng/mL had no effect on T cell activation, suggesting that the common sCD38 concentration in patients was insufficient to trigger Ab-A-mediated T cell activation.

In the presence of CD38-positive target cells, Ab-A induces T cells to produce proinflammatory interleukins, including TNF-α and IFN-γ. Evaluate the effect of dexamethasone on Ab-A activity. Human T cells were co-cultured with KMS-12-BM luc MM target cells at an E:T ratio of 1 in the presence of dexamethasone and increasing concentrations of Ab-A. Pretreatment effect of dexamethasone with a gradually decreasing concentration (starting at 230 ng/mL, ending at 24 hours and ending at 12.6 ng/mL, which is equivalent to the serum concentration in human subjects at 24 hours after a 20 mg oral dexamethasone dose) Cells and target cells. The redirected lysis assay was then performed with Ab-A at an E:T ratio of 1 at a continuous concentration of 12.6 ng/mL dexamethasone. Under these clinically relevant conditions, the killing curves of KMS-12-BM luc MM target cells in the presence or absence of dexamethasone look very similar. When the E:T ratio is 1, the Ab-A target cleavage EC50 increases less than 2-fold, but the levels of IFN-γ and TNF-α cytokines decrease by more than 85%. See Figure 21. The data shows that Ab-A can be administered 24 hours after oral administration of dexamethasone, which has a limited impact on the efficacy of Ab-A, but has the potential benefit of lower cytokine release.

The in vivo efficacy of Ab-A is supported by data showing its antitumor effect in a MOLM-13 luc orthotopic mouse xenograft model supplemented with human T cells. Treatment with Ab-A significantly prolonged the survival of mice carrying established MOLM-13 luc tumors in situ. NOD Scidγ (NSG) immunocompromised mice were orthotopically transplanted with MOLM-13 luc tumor cells expressing CD38 and injected intraperitoneally with human T cells 2 days later. Ab-A was administered at 0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg by intravenous bolus every 7 days from day 5 for 35 days. The survival data is presented graphically in the Kaplan-Meier chart. Compared with vehicle-treated control group (22.0 days), Ab-A administration resulted in prolonged median survival (37.5 days and 36 days at 0.01 mg/kg, 0.1 mg/kg, and 1 mg/kg dose levels, respectively) And 37 days). The comparison of survival between groups (using the log rank test for the vector as a control) demonstrated that Ab-A-induced prolongation of survival was highly statistically significant (p <0.0001). Note the lack of dose response because the maximum effect is observed at the lowest dose of 0.01 mg/kg. Whole bioluminescence (BLI) imaging was used to monitor tumor growth. One week after the first dose was administered on Day 4, compared to vehicle control animals, representative BLI images showed that tumors in the hindlimb and spine areas were clearly eliminated in all treatment groups. By day 22, tumor growth inhibition (TGI) at the dose levels of 0.01 mg/kg, 0.1 mg/kg, and 1.0 mg/kg Ab-A was 98%, 93%, and 99%, respectively, and no significant weight loss was observed .

In addition, Ab-A was shown to have pharmacological activity in vivo in cynomolgus monkeys, as shown by Ab-A-induced depletion of peripheral B cells, activation of T cells, and depletion of peripheral T cells. Ab-A triggers B cell depletion in vitro in an auto-redirected lysis assay using cynomolgus monkey PBMC. In order to monitor the in vivo pharmacodynamics of Ab-A, a single dose of 10 μg/kg on day 1 (n = 3/group) was used at 150 μg/kg on day 2, day 5, day 8, and day 11 After intravenous bolus injection, the fate of peripheral B cells was also evaluated in cynomolgus monkeys. A decrease in peripheral B cells was observed after the first dose was administered, and the level returned to baseline before the dose was administered on the fifth day. By day 11, a significant average reduction of 95.5% of peripheral B-cell numbers compared to pre-dose levels was observed. In 1 animal, the number of B cells recovered after day 11 and this recovery was accompanied by a decrease in Ab-A serum levels. In the second cynomolgus monkey study of Ab-A, a dose-dependent transient decrease in the number of peripheral B cells and T cell activation on day 4 was also observed, confirming that Ab-A has in vivo activity in cynomolgus monkeys and triggers T cells Activation and CD38-positive cell depletion are both expected results of the mechanism of Ab-A cell recruitment. In cynomolgus monkey toxicology studies, Ab-A does not affect neurological, respiratory, or cardiovascular safety pharmacological parameters. Performance analysis identified the strongest expression of CD38 in hematopoietic/lymphoid cells and tissue lines, and the safety assessment of cynomolgus monkeys was consistent with this performance pattern.

Demonstrated the efficacy and specificity of the Ab-A a number of studies in vitro and in vivo, cynomolgus and related species has proven to be used to assess safety. Clinical research

Subjects will participate in a dose-exploration cohort, using Bayesian logistic regression model (BLRM) to evaluate the maximum tolerated dose (MTD), safety, and safety of different doses of Ab-A in patients with relapsed or refractory multiple myeloma Tolerance, PK and PD. Ab-A will initially be administered as an intravenous infusion as follows: twice a week for weeks 1 and 2, once a week for weeks 3 and 4, once every other week for weeks 5 and thereafter. Alternative dosing regimens are as follows: once a week in week 1, week 2, week 3 and week 4, and every other week in week 5 and thereafter. The planned dose levels (dose per infusion) of the dose exploration group are as follows: 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg, and 200 mg. Unless there are contraindications for pre-administration, pre-administer IV dexamethasone within 1 hour before the start of Ab-A; the initial pre-administration will contain 8 mg dexamethasone, if the treatment is well tolerated, the dose will be reduced To 4 mg.

Various parameters are measured during treatment, including vital signs, liver chemistry, hematological parameters, renal function and related adverse events (AE). Evaluation of minimal residual disease (MRD) is also possible. MRD evaluation can utilize, for example, next generation sequencing (NGS) of samples from bone marrow and/or blood and/or flow cytometry using bone marrow. Evaluation can be performed before treatment (baseline), after one or more rounds of treatment, and/or after the IMWG guidelines are followed to determine complete remission. Cytokine Release Syndrome (CRS) (excessive production of cytokines associated with CD3 conjugation) and fever, chills, fatigue, fatigue, headache, mental state changes, speech disorders, tremor, difficulty in distinguishing distance, abnormal gait, Seizures, dyspnea, shortness of breath, hypoxemia, tachycardia, hypotension, nausea, vomiting, elevated transaminases, hyperbilirubinemia, bleeding, hypofibrinogenemia, D-di Elevated aggregates are associated with rashes. These symptoms will be monitored. The CRS ratings are as follows: Level 1, the symptoms are not life-threatening and only require symptomatic treatment (such as fever, nausea, fatigue, headache, myalgia, and fatigue); Level 2, the symptoms require and moderate intervention for adaptation (oxygen demand <40% , Or hypotension in response to fluids or low doses of a vasopressor, or grade 2 organ toxicity or grade 3 transaminase elevation meets CTCAE standards); grade 3, symptoms require concurrent active intervention (oxygen demand ≥ 40%, or hypotension requires high doses or multiple vasopressors, or grade 3 organ toxicity or grade 4 transaminase hypotension/CTCAE standard) response; and grade 4, life-threatening symptoms (according to CTCAE standard) Requires ventilator support or grade 4 organ toxicity (excluding elevated transaminase)). In various aspects, the subject experienced little or no symptoms of CRS (eg, the subject experienced grade 2, grade 1, or no CRS.

Determine the overall response rate (ORR) according to the International Myeloma Working Group (IMWG) response criteria (strict [s] complete response [CR], CR, very good [VG] partial response [PR], PR) The methods of response duration, progression time, progression-free survival and overall survival are known in the art. A complete response (CR) is characterized by, for example, negative immunofixation to serum and urine and the disappearance of any soft tissue plasmacytoma and <5% plasma cells in the bone marrow. The strict complete response is characterized by the CR as defined above and the normal FLC ratio and the absence of colonized cells in the bone marrow by immunohistochemistry or immunofluorescence. VGPR is characterized by, for example, the detection of serum and urinary M proteins by immunofixation, but not by electrophoresis, or a reduction of serum M protein >90% plus urine M protein levels <100 mg/24 h. PR is characterized by, for example, a reduction in serum M protein >50% and a 24-hour reduction in urine M protein >90% or <200 mg/24 h; if serum and urine M proteins are not measurable, then FLC levels that are involved and unaffected are required The difference between them is reduced by >50% to replace the M protein standard; if serum and urine M proteins are not measurable, and serum free photometry is not measurable, then a >50% reduction in plasma cells is required to replace the M protein, the condition is baseline bone marrow The percentage of plasma cells is> 30%; if present at baseline, the size of the soft tissue plasma cell tumor is reduced by> 50%. See, for example, the International Myeloma Working Group (IMWG) unified response criteria for multiple myeloma can be obtained at: imwg.myeloma.org/international-myeloma-working-group-imwg-uniform-response-criteria-for- multiple-myeloma/. The present disclosure considers improvement of any of these parameters, and preferably improvement sufficient to achieve at least PR, at least VGPR, CR, or sCR.

[Figure 1A] and [1B] depict several forms of heterodimeric antibodies. Two forms of "bottle opener" versions are depicted, one with an anti-CD3 antigen-binding domain containing scFv and an anti-CD38 antigen-binding domain containing Fab, and the other with these reversed. All mAb-Fv, mAb-scFv, center scFv and center Fv types are shown. In addition, the "single-arm" version in which one of the monomers contains only the Fc domain is shown as both the single-arm center scFv and the single-arm center Fv. The double scFv version is also shown.

[Figure 2] depicts the sequence of the "高-中#1" anti-CD3_H1.32_L1.47 construct, including the variable heavy and light chain domains (CDR plus bottom line), as well as the separate vl and vhCDR, and the band The scFv construct of the charge linker (plus double bottom line). As with all sequences depicted in the figure, this charged linker can be replaced with an uncharged linker or a different charged linker, as needed.

[Figure 3] depicts the sequence of the CD38: OKT10_H1L1.24 construct, including variable heavy and light chain domains (CDR plus bottom line), as well as individual vl and vhCDR, and a charged linker (double bottom line ) ScFv construct.

[Figure 4] depicts the sequence of the low CD38: OKT10_H1L1 construct, including the variable heavy and light chain domains (CDR plus bottom line), as well as the individual vl and vhCDR, and the charged linker (plus double bottom line) The scFv construct.

[Figure 5] depicts the sequence of XENP18971.

[Figure 6] depicts the sequence of XENP18969.

[Figure 7] depicts the sequence of human CD3 epsilon (SEQ ID NO: 130).

[FIG. 8] Depicts the full-length of human CD38 protein (SEQ ID NO: 131) and extracellular domain (ECD; SEQ ID NO: 132).

[Figures 9A-9E] depict useful pairs of heterodimerization variants (including skew variants and pI variants).

[Fig. 10] A list of isosteric variant antibody constant regions and their respective substitutions is depicted. pI_(-) indicates a lower pI variant, and pI_(+) indicates a higher pI variant. These can be combined with other heterodimerization variants of the invention (as well as other variant types as outlined herein) as needed and independently.

[Figure 11] Depicts useful ablation variants of ablation of Fc[gamma]R binding (sometimes referred to as "knockout" or "KO" variants).

[Fig. 12] Two embodiments of the present invention are shown.

[FIG. 13A] and [13B] depict many charged scFv linkers, which can be used to increase or decrease the pi of a heterodimeric antibody using one or more scFv as a component. A single prior art scFv linker with a single charge is called "Whitlow" from Whitlow et al., Protein Engineering [Protein Engineering] 6(8): 989-995 (1993). It should be noted that this linker is used to reduce aggregation in scFv and enhance proteolytic stability.

[Fig. 14] A list of engineered heterodimer-skew Fc variants, together with heterodimer yield (determined by HPLC-CIEX) and thermal stability (determined by DSC) is depicted. Undetermined thermal stability is indicated by "n.d".

[Figures 15A-15B] Depicted stability optimized humanized anti-CD3 variant scFv. The substitution is given relative to the H1_L1.4 scFv sequence. The amino acid number is the Kabat number.

[Figures 16A-16B] Depicts the amino acid sequence of stability optimized humanized anti-CD3 variant scFv. CDR plus bottom line. For each heavy/light chain combination, four sequences are listed: (i) scFv with C-terminal 6xHis tag, (ii) scFv only, (iii) VH only, (iv) VL only.

[Figure 17] depicts the sequence of XENP18971.

[Figure 18] depicts the sequence of XENP18969.

[Fig. 19] A matrix showing possible combinations of embodiments. "A" means that the CDR of the cited CD3 sequence can be combined with the CDR of the CD38 construct on the left. That is, for example, for the cell in the upper left corner, the vhCDR from the variable heavy chain CD3 H1.30 sequence and the vlCDR from the variable light chain of CD3 L1.47 sequence can be compared with the vhCDR from CD38 OKT10 H1.77 sequence Combined with vlCDR from OKT10L1.24 sequence. "B" means that the CDR from the CD3 construct can be combined with the variable heavy and light chain domains from the CD38 construct. That is, for example, for the upper left cell, the vhCDR from the variable heavy chain CD3 H1.30 sequence and the vlCDR from the variable light chain of CD3 L1.47 sequence can be combined with the variable heavy chain domain CD38 OKT10 H1.77 sequence Combination with OKT10L1.24 sequence. Reverse "C" so that the variable heavy chain domain and variable light chain domain from the CD3 sequence are used with the CDR of the CD38 sequence. "D" comes from the combination of each variable heavy chain and variable light chain. "E" refers to the case where the scFv of CD3 is used with the CDR of the CD38 antigen-binding domain construct, and "F" the scFv of CD3 is used with the variable heavy and variable light chain domains of the CD38 antigen-binding domain Case.

[Fig. 20] depicts the sequence of Ab-A.

[FIG. 21] Includes two graphs illustrating the response of dexamethasone pretreatment and then exposure to Ab-A described in Examples. The left graph correlates cytotoxicity (%) (Y axis) with Ab-A concentration (ng/ml). Pretreatment with dexamethasone has minimal effect on the EC50 of the heterodimeric antibody. The right panel correlates cytokine production (IFNγ, pg/ml) (y-axis) with Ab-A concentration (ng/ml). Pretreatment resulted in an 85% reduction in cytokine release.

[Fig. 22] A table that associates each CDR sequence, variable region sequence, heavy and light chain sequence, scFv sequence, main chain sequence, etc. with the sequence identifiers listed in the sequence table attached to this application. Regarding the main chains of the several types of corkscrew mentioned (SEQ ID NO: 347-354), Fv sequences of such sequences are not provided (eg, scFv and vh and vl on the Fab side). As understood by those skilled in the art and outlined below, these sequences can be used with any of the vh and vl pairs outlined herein, where one monomer includes scFv (including charged scFv linkers as needed) and the other Fab sequences are expanded in body (eg, vh attached to the "Fab side heavy chain" and vl attached to the "constant light chain"). The scFv can be anti-CD3 or anti-CD38, another line of Fab. ("Fab" refers to the portion comprising the immunoglobulin domains of VH, CH1, VL, and CL.) That is, any Fv sequence of CD3 and CD38 outlined herein can be incorporated into these backbones in any combination.

Claims (38)

A method for treating multiple myeloma, the method comprising administering a heterodimeric antibody to a subject in need at a dose of about 0.05 mg to about 200 mg, the heterodimeric antibody comprising: a) a first monomer, the first monomer comprising a first Fc domain and an anti-CD3 scFv, the anti-CD3 scFv comprising (i) scFv variable light chain domain comprising vlCDR1 as shown in SEQ ID NO: 15, vlCDR2 as shown in SEQ ID NO: 16, and vlCDR3 as shown in SEQ ID NO: 17, and (ii) a scFv variable heavy chain domain comprising vhCDR1 shown in SEQ ID NO: 11, vhCDR2 shown in SEQ ID NO: 12, and vhCDR3 shown in SEQ ID NO: 13, wherein the structure is used A domain linker covalently attaches the scFv to the N-terminus of the Fc domain; b) Second monomer, the second monomer contains i) an anti-CD38 heavy chain variable domain comprising vhCDR1 as shown in SEQ ID NO: 65, vhCDR2 as shown in SEQ ID NO: 66, and vhCDR3 as shown in SEQ ID NO: 67, and ii) a heavy chain constant domain comprising a second Fc domain; and c) A light chain comprising a variable constant domain and an anti-CD38 variable light chain domain, the anti-CD38 variable light chain domain comprising vlCDR1 as shown in SEQ ID NO: 69, as shown in SEQ ID NO: 70 VlCDR2, and vlCDR3 shown in SEQ ID NO: 71. The method of claim 1, wherein the dose is about 0.05 mg, 0.15 mg, 0.45 mg, 1.35 mg, 4 mg, 12 mg, 36 mg, 100 mg, or 200 mg. The method of claim 1, wherein the dose is about 36 mg to about 200 mg. The method according to any one of claims 1 to 3, the method comprising: administering two doses of heterodimeric antibody to the subject each week during the first week and the second week of treatment. The subject was administered a dose of heterodimeric antibody weekly for three weeks and the fourth week, and a dose of heterodimeric antibody was administered every two weeks from week 5 to the end of treatment. The method of any one of claims 1 to 3, the method comprising: administering a dose of heterodimerization to the subject weekly during the first week, second week, third week, and fourth week of treatment Antibodies, and a dose of heterodimeric antibody is administered every two weeks from the 5th week to the end of treatment. The method of any one of claims 1 to 5, wherein the method comprises administering two or more doses of the heterodimeric antibody during a treatment period of about 6 months to about 12 months. The method of claim 6, wherein the treatment period is about eight months. The method of any one of claims 1 to 7, wherein the heterodimeric antibody is administered by intravenous infusion over about 30 minutes to about 4 hours. The method of claim 8, wherein the first dose of heterodimeric antibody is administered in about four hours, the second dose of heterodimeric antibody is administered in about two hours, and all subsequent doses are administered in about Give in 30 minutes. The method of any one of claims 1 to 9, the method comprising: administering two or more doses of heterodimeric antibody and increasing the dose at least once during the treatment period. The method of any one of claims 1 to 10, wherein the subject has relapsed/refractory multiple myeloma. The method of any one of claims 1 to 11, the method further comprising administering dexamethasone to the subject. The method of claim 12, wherein the dexamethasone is administered intravenously. The method of claim 12 or claim 13, wherein the dexamethasone is administered to the subject within one hour before the administration of the heterodimeric antibody. The method of any one of claims 12 to 14, wherein dexamethasone is administered in an amount of about 8 mg or about 4 mg. The method of claim 12, wherein dexamethasone is administered orally. The method of any one of claims 1 to 16, the method comprising: (a) administering an anti-CD38 monospecific antibody to the subject, and reducing the systemic concentration of the anti-CD38 monospecific antibody to a level sufficient to After an elution period of 0.2 μg/ml or less, (b) administer the initial dose of the heterodimeric antibody. The method of any one of claims 1 to 16, the method comprising: (a) administering the anti-CD38 monospecific antibody to the subject and (b) at least after stopping the administration of the anti-CD38 monospecific antibody The initial dose of this heterodimeric antibody was administered at the 12-week time point. The method of claim 18, wherein the method comprises stopping treatment of the anti-CD38 monospecific antibody 14-16 weeks prior to the administration of the initial dose of the heterodimeric antibody. The method of any one of claims 17 to 19, wherein the anti-CD38 monospecific antibody system is daratumumab. The method of any one of claims 1 to 20, wherein the subject has previously received treatment with a proteasome inhibitor and/or immunomodulatory drug. The method of any one of claims 1 to 21, wherein the anti-CD3 scFv comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 18. The method of any one of claims 1 to 21, wherein the anti-CD3 scFv comprises the amino acid sequence shown in SEQ ID NO: 18. The method of any one of claims 1 to 23, the anti-CD38 variable light chain domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 68. As in the method of claim 24, the anti-CD38 variable chain light chain domain comprises the amino acid sequence shown in SEQ ID NO: 68. The method of any one of claims 1 to 25, wherein the anti-CD38 heavy chain variable domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 64. The method of claim 26, wherein the anti-CD38 heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 64. The method of any one of claims 1 to 27, wherein the first monomer comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 335. The method of claim 28, wherein the first monomer comprises the amino acid sequence shown in SEQ ID NO: 335. The method of any one of claims 1 to 29, wherein the second monomer comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 82. The method of claim 30, wherein the second monomer comprises the amino acid sequence shown in SEQ ID NO: 82. The method of any one of claims 1 to 31, wherein the light chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 84. The method of claim 32, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 84. The method of any one of claims 1 to 27, wherein the first Fc domain and the second Fc domain comprise one or more mutations that reduce homodimerization. The method according to any one of claims 1 to 27, wherein the first Fc domain and the second Fc domain comprise a variant group selected from the group consisting of the following items: S364K/ E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q. The method according to any one of claims 1 to 27, wherein the scFv domain linker is a charged linker. The method of any one of claims 1 to 27, wherein the heavy chain constant domain comprises an amino acid substitution N208D/Q295E/N384D/Q418E/N421D. The method of any one of claims 1 to 27, wherein the first and second Fc domains comprise amino acid substitutions E233P/L234V/L235A/G236del/S267K.

Images