How to Treat Non-Hodgkin's Lymphoma

Abstract

Methods of treating non-Hodgkin's lymphoma by administering multispecific antibodies to a patient in need thereof are provided. Methods of making such antibodies and compositions, including pharmaceutical compositions, comprising such antibodies are also provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Nos. 63 / 381,886, filed November 1, 2022, and 63 / 504,179, filed May 24, 2023, both of which are incorporated by reference in their entireties for all purposes.

[0002] (Reference to sequence listing) This application incorporates by reference the Sequence Listing submitted herewith as an HTML file with the name CD19TCE-201-WO-PCT, created October 31, 2023, and size 28.0 bytes.

[0003] FIELD OF THE INVENTION The present disclosure provides methods of treating non-Hodgkin's lymphoma by administering multispecific (e.g., bispecific) antibodies to a patient in need thereof. The present disclosure further provides methods of making such antibodies, and compositions, including pharmaceutical compositions, comprising such antibodies. [Background technology]

[0004] The annual rate of new non-Hodgkin lymphoma (NHL) cases was 19.6 per 100,000 men and women. The annual mortality rate was 5.4 per 100,000 men and women. These rates are age-adjusted and based on cases and deaths from 2014 to 2018. The estimated new cases in 2021 are 81,560, representing 4.3% of all new cancer cases. The estimated new deaths in 2021 are 20,720, representing 3.4% of all cancer deaths. At initial diagnosis, 24% of cases were stage I, defined as "confined to a single area," 14% were stage II, defined as "involving multiple areas," 16% were stage III, defined as "spread to both sides of the diaphragm," 33% were stage IV, defined as "diffuse or disseminated disease," and 12% were unstaged. The 5-year relative survival rates were 84.3% for stage I, 77.1% for stage II, 71.1% for stage III, and 63.7% for stage IV. SEER Database, accessed April 15, 2021, https: / / seer.cancer.gov / .

[0005] Non-Hodgkin lymphomas comprise a diverse range of neoplasms of the lymphoid compartment, with approximately 74,000 new cases reported annually (National Cancer Institute [NCI] Surveillance, Epidemiology and End Results [SEER] 2020). In the United States (US), an estimated 80% of these are of the B-cell lineage. Within B-cell non-Hodgkin lymphoma (B-NHL), subtypes can be broadly categorized into “indolent” lymphomas, including chronic lymphocytic leukemia / small lymphocytic lymphoma (not investigated in this trial), follicular lymphoma (FL), and marginal zone lymphoma (MZL), and “aggressive” lymphomas, including diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL), mantle-cell lymphoma (MCL), and transformed indolent lymphoma, with additional types and subtypes existing (Swerdlow 2017).

[0006] Diagnosis often involves techniques such as physical examination, blood and urine tests, imaging, and lymph or bone marrow tests. Traditional therapies include chemotherapy administered orally or by injection, radiation therapy, and bone marrow transplantation.

[0007] For DLBCL, more than half of patients survive for more than 5 years using multiagent chemotherapy regimens including rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (e.g., R-CHOP; Friedberg 2011). Second-line therapy typically consists of high-dose chemotherapy followed by autologous SCT. For transplant-ineligible patients, broad-spectrum therapies, usually incorporating anti-CD20 monoclonal antibodies, are used. Of these, only 30–40% respond (Crump 2017). The prognosis for patients who do not respond to salvage therapy is poor. Third-line therapies include anti-CD19 chimeric antibody receptor (CAR) T cells (e.g., Yescarta® and Kymriah®) and the anti-CD79b ADC Polivy® (Yescarta US Prescribing Information [PI] 2017; Kymriah US PI 2017; Polivy US PI 2019). Eligibility for second- and third-line therapies varies greatly among patients. For example, elderly or frail patients are often not transplant candidates, while up to 23% of patients with relapsed or refractory (RR) DLBCL have disease that progresses too rapidly to qualify for CAR T-cell therapy (Paillassa 2019). Furthermore, all of the above-mentioned third-line therapies are not currently considered "available therapies." Beyond second-line treatment, overall survival (OS) ranges from 8 to 10 months (Van Den Neste 2016). Given these characteristics, DLBCL, particularly RR or high-risk disease, represents one of the most important areas of unmet medical need in lymphoma.

[0008] Aspects of the present disclosure include methods of treating this patient population. Summary of the Invention

[0009] Embodiments of the present disclosure include a method for treating B-cell non-Hodgkin's lymphoma in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to 28-day treatment cycles, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg, and optionally, the patient receives six treatment cycles.

[0010] In some embodiments, the treatment cycle is repeated two or more times. In some embodiments, TNB-486 is administered to the patient as monotherapy. In some embodiments, TNB-486 is administered by intravenous infusion (IV). In some embodiments, the patient has received at least two prior line of systemic therapies. In some embodiments, the patient is CD19-positive. In some embodiments, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less. In some embodiments, the patient has adequate bone marrow function. In some embodiments, the patient has an estimated glomerular filtration rate (eGFR) of 50 mL / min or greater. In some embodiments, the patient has a total bilirubin level of 1.5 times the upper limit of normal, an aspartate aminotransferase (AST) level of 3 times the upper limit of normal, and an alanine aminotransferase (ALT) level of 3 times the upper limit of normal.

[0011] Embodiments of the present disclosure include a method for improving the objective response rate (ORR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0012] Embodiments of the present disclosure include a method for improving overall survival (OS) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0013] Embodiments of the present disclosure include a method for improving progression-free survival (PFS) rates in patients diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0014] Embodiments of the present disclosure include a method for improving time to progression (TTP) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0015] Embodiments of the present disclosure include a method for improving time to response (TTR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0016] Embodiments of the present disclosure include a method for improving duration of objective response (DOR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0017] Embodiments of the present disclosure include a method for improving clinical benefit rate (CBR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, the method comprising administering to the patient a therapeutically effective amount of TNB-486 according to a 28-day cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0018] In some embodiments, the improvement is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

[0019] In some embodiments, the treatment cycle is modified to add more time between doses. In some embodiments, the treatment cycle is modified by consistently eliminating one or more treatment cycles from the dosing regimen.

[0020] In some embodiments, the treatment cycle is modified to include a priming dose. In some embodiments, the priming dose is from about 150 μg to about 1500 μg. In some embodiments, the priming dose is from about 270 μg to about 1000 μg. In some embodiments, the priming dose is administered at the first time point in the first treatment cycle, and a full dose is administered at all subsequent time points. In some embodiments, the priming dose is administered on day 1 of the first treatment cycle, a full dose is administered on day 15 of the first treatment cycle, and a full dose is administered on days 1 and 15 of all subsequent treatment cycles.

[0021] In some embodiments, the treatment cycle is modified to include at least two priming doses. In some embodiments, the first priming dose is about 150 μg to about 540 μg. In some embodiments, the second priming dose is about 800 μg to about 1200 μg. In some embodiments, the first priming dose is about 270 μg and the second priming dose is about 1000 μg. In some embodiments, the first priming dose is administered on day 1 of the first treatment cycle, the second priming dose is administered on day 8 of the first treatment cycle, a complete dose is administered on day 15 of the first treatment cycle, and a complete dose is administered on days 1 and 15 of all subsequent treatment cycles.

[0022] In some embodiments, the full dose is no more than 100% greater (2-fold greater) than the full dose corresponding to the next lowest dose cohort. In some embodiments, the full dose is no more than 50% greater than the full dose corresponding to the next lowest dose cohort. In some embodiments, the full dose is no more than 33% greater than the full dose corresponding to the next lowest dose cohort.

[0023] In some embodiments, the method further comprises pre-medicating the patient with an agent that reduces the risk or severity of a hypersensitivity reaction prior to administration of TNB-486. In some embodiments, the agent that reduces the risk or severity of a hypersensitivity reaction is selected from the group consisting of dexamethasone, diphenhydramine, acetaminophen, ranitidine, tocilizumab, any equivalent thereof, or any combination thereof. In some embodiments, the agent that reduces the risk or severity of a hypersensitivity reaction is administered 15 to 60 minutes prior to administration of TNB-486. In some embodiments, the therapeutically effective amount of TNB-486 is about 30 μg, 90 μg, 270 μg, 800 μg, 2400 μg, 7200 μg, 15000 μg, or 30000 μg.

[0024] In some embodiments of the method, TNB-486 is administered in combination with another chemotherapy. In some embodiments, the other chemotherapy is a combination of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunomycin), vincristine sulfate (Oncovin), and prednisone (R-CHOP). In some embodiments, R-CHOP is administered on day 1 of the first treatment cycle, and then on day 1 of each subsequent treatment cycle. In some embodiments, each treatment cycle is 21 days long, R-CHOP is administered on day 1 of the first treatment cycle, a first priming dose is administered on day 8 of the first treatment cycle, a second priming dose is administered on day 15 of the first treatment cycle, and R-CHOP and a therapeutically effective amount of TNB-486 are administered on day 1 of the second treatment cycle. In some embodiments, R-CHOP and a therapeutically effective amount of TNB-486 are administered on day 1 of each subsequent treatment cycle.

[0025] In some embodiments, the patient experiences reduced cytokine release compared to a different TNB-486 dosing schedule. In some embodiments, the patient does not experience cytokine release syndrome or only experiences Grade 1 cytokine release syndrome. In some embodiments, the cytokine release syndrome comprises release of IL-6 and / or TNF-α. In some embodiments, the objective response rate (ORR) is 80% or greater. In some embodiments, the complete response (CR) rate is 90% or greater. In some embodiments, the 6-month PFS rate is 90% or greater. The method of any preceding claim, wherein TNB-486 is a bispecific molecule that binds to CD3 and human CD19 and comprises: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 18; (ii) a second polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 11 (the first polypeptide subunit and the second polypeptide subunit together form a first binding moiety that binds human CD3); and (iii) a third polypeptide subunit that binds human CD19 comprising the amino acid sequence of SEQ ID NO: 20.

[0026] These and further aspects are further described in the remainder of the disclosure, including the Examples. [Brief explanation of the drawings]

[0027] [Figure 1A] FIG. 1 is a schematic diagram illustrating an exemplary treatment method including a dose escalation phase (Arm A) and two dose expansion phases (Arm B and Arm C). [Figure 1B] FIG. 1 is a schematic diagram showing an exemplary treatment method involving administration of TNB-486 in combination with R-CHOP (Arm D). [Figure 2] FIG. 1 is a schematic diagram showing an exemplary treatment method including a dose escalation phase (Arm A) and providing additional details regarding the treatment of different patient cohorts. [Figure 3-1] FIG. 1 is a schematic diagram showing suggested treatment guidelines for subjects exhibiting signs and / or symptoms of cytokine release syndrome (CRS). [Figure 3-2] FIG. 1 is a schematic diagram showing suggested treatment guidelines for subjects exhibiting signs and / or symptoms of cytokine release syndrome (CRS). [Figure 4] FIG. 1 is a schematic diagram of TNB-486, a three chain antibody-like molecule (TCA) containing one arm containing a heavy / light chain pair that binds to CD3 in a monovalent configuration and a second arm containing the variable region of only the heavy chain that binds to CD19. [Figure 5A] 1 is a plot of the incidence of cytokine release syndrome at different time points of administration as described in the Examples. C1D1 - Cycle 1, Day 1. C1D15 - Cycle 1, Day 15. C2D1 - Cycle 2, Day 1. The total incidence of CRS is indicated above each bar. [Figure 5B]1 is a plot of the incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) at different time points of administration as described in the Examples. C1D1 - cycle 1, day 1. C1D15 - cycle 1, day 15. C2D1 - cycle 2, day 1. The total incidence of ICANS is indicated above each bar. [Figure 6] 1 is a schematic diagram of patient outcomes for subjects assessed at the first clinical cutoff described in the Examples. Lymphoma type is as indicated in the key. CR - complete response. DC - PD - discontinued, patient decision. DC - other - discontinued, other reason. PR - partial response. [Figure 7] 1 shows plots of response rates for the first clinical cutoff described in the Examples. The first plot is for all types of B-cell Hodgkin's lymphoma (B-NHL) subjects (n=25). The second plot is for four subjects with diffuse large B-cell lymphoma (DLBCL). The third plot is for eight subjects with follicular lymphoma (FL). The overall response rate (ORR) is shown above the first and second plots, and the complete response (CR) rate is shown in the third plot. [Figure 8] 1 is a schematic diagram of patient outcomes for subjects assessed at the second clinical cutoff described in the Examples. Lymphoma types are as indicated in the key. CR - complete response. DC - PD - discontinued, patient decision. DC - other - discontinued, other reason. PR - partial response. [Figure 9] Median cytokine levels (pg / ml) of both IL-6 and TNF-α for the various preparative dose regimens are shown. [Figure 10] Complete response (CR) is typically achieved at the first evaluation (week 8), and the 6-month PFS rate is 91%. DETAILED DESCRIPTION OF THE INVENTION

[0028] The practice of the present methods will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are described in "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RIFreshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), and "Current Protocols in Molecular Biology" (FM Ausubel et al. al., eds., 1987, and periodic revisions), “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), “Phage Display: A Laboratory Manual” (Barbas et al., 2001), Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No.I, Cold Spring Harbor Laboratory (1998), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0029] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0030] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0031] In the following description, numerous specific details are set forth to provide a more thorough understanding of the methods disclosed herein. However, it will be apparent to one skilled in the art that the methods may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art are not described to avoid obscuring the methods disclosed herein.

[0032] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0033] I. Definition "Comprising" means that the stated element is required for the composition / method / kit, but that other elements may be included to form a composition / method / kit, etc. within the scope of the claim.

[0034] "Consisting essentially of" means limiting the scope of the described composition or method to those specified materials or steps that do not materially affect the basic and novel characteristics of the method.

[0035] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim.

[0036] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (around residues 1 to 113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as per Kabat" refers to the residue numbering of a human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0037] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain; the amino-terminal domains of the heavy and light chains are variable in sequence and are therefore commonly referred to as variable region domains, or variable heavy (VH) or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be achieved with variable sequence in the heavy chain alone, and various non-natural configurations of antibodies are known and used in the art.

[0038] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy-chain-only antibodies and multispecific (e.g., bispecific) three-chain antibody-like molecules (TCAs) described herein) is one that can exert one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, such as a TCA, may have the ability to specifically bind to an antigen, which binding can in turn trigger or modify a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as a TCA, to exert one or more of its native activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0039] The term "adverse event" or "AE" is defined as any untoward medical occurrence in a clinical trial subject administered a medicinal product, which does not necessarily have a causal relationship to treatment. Thus, an AE can be any untoward and unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of a medicinal product (investigational drug), regardless of whether the event is considered causally related to the use of the medicinal product.

[0040] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fvs (scFvs), nanobodies, and the like, as well as antibody fragments so long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0041] The term "antibody" may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds an antigen or portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule (including engineered subclasses with modified Fc portions that result in reduced or enhanced effector cell activity). The light chain of a subject antibody can be a kappa light chain (Vkappa) or a lambda light chain (Vlambda). The immunoglobulin can be derived from any species. In one aspect, the immunoglobulin is predominantly human in origin.

[0042] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies in accordance with the methods of the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced via recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0043] The term "variable" as used in reference to antibodies refers to the fact that certain portions of antibody variable domains differ widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FRs, which are predominantly in a β-sheet configuration connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet configuration. The hypervariable regions of each chain are held in close proximity by FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

[0044] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some aspects, "CDR" refers to the complementarity determining regions of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0045] While exemplary CDR designations are provided herein, those skilled in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition, which is based on sequence variability and is the most commonly used (see, "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700). The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include, but are not limited to, those described in Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001;309:657-670; Ofran et al., "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes." J Immunol. 2008;181:6230-6235; Almagro, "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004;17:132-143; and Padlan et al., "Identification of specificity-determining residues in antibodies." Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0046] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer in the broadest sense to an antibody or one or more portions of an antibody, e.g., one or more arms, that lack the light chain of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains, but no CH1 domain, functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NARs), and functional fragments thereof, and soluble single-domain antibodies (sUniDabs™). In one aspect, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains have been truncated are also included herein. In a further embodiment, the heavy chain is composed of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but does not include the hinge region. Heavy chain-only antibodies may be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or non-covalently bound to each other. Heavy chain-only antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In particular embodiments, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, the heavy chain-only antibodies herein are used as the binding (targeting) domain of a chimeric antigen receptor (CAR).This definition specifically includes human heavy chain-only antibodies produced by human immunoglobulin transgenic rats (UniRat™), which are referred to as UniAbs™. The variable regions (VH) of UniAbs™, called UniDabs™, are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multispecificity, increased potency, and extended half-life. Because homodimeric UniAbs™ lack light chains and therefore VL domains, antigens are recognized by one single domain, i.e., the variable domain of the heavy chain of a heavy chain antibody (VH or VHH).

[0047] As used herein, an "intact antibody chain" comprises a full-length variable region and a full-length constant region (Fc). An intact, "traditional" antibody comprises an intact light chain and an intact heavy chain, as well as the light chain constant domain (CL) and heavy chain constant domains of secreted IgG, CH1, hinge, CH2, and CH3. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (native-sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors. Constant region variants include those that alter effector profile, Fc receptor binding, etc.

[0048] Antibodies and various antigen-binding proteins can be provided as different classes depending on the amino acid sequence of the Fc (constant domain) of their heavy chains. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies may be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; U.S. Patent Application Publication Nos. 2005 / 0048572 and 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains. Antibodies according to embodiments of the present methods can comprise a kappa light chain sequence or a lambda light chain sequence.

[0049] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally involve the ability of an Fc region to bind to receptors, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, which can be assessed using various assays known in the art. A "dead" or "silenced" Fc is one that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0050] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include, for example, native-sequence human IgG1 Fc regions (non-A allotypes and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0051] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably about one to about five amino acid substitutions. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0052] The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is incorporated herein by reference. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4):pp.915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0053] Other Fc variants are possible, including, but not limited to, those in which regions capable of forming disulfide bonds are deleted, or certain amino acid residues are removed at the N-terminus of a native Fc, or a methionine residue is added. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet other embodiments, the hinge region of the Fc may be removed entirely. In yet other embodiments, an antibody may comprise an Fc variant.

[0054] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, but not limited to, deletions may occur in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0055] In some aspects, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some aspects, the antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner to place a "knob" on the first heavy chain constant region of a first monomer in an antibody dimer and a "hole" on the second heavy chain constant region of a second monomer in the antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0056] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P, F234A, L235A, and T366W mutation (knob). In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P, F234A, L235A, T366S, L368A, and Y407V mutation (hole).

[0057] The term "Fc region-containing antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of a nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present disclosure can include antibodies with or without K447.

[0058] Embodiments of the present disclosure include antibodies comprising heavy chain-only variable regions in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" when used with respect to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, having a single binding site. In contrast, the term "bivalent configuration" when used with respect to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (each having a single binding site) and are connected by a linker sequence (see Figure 4). Non-limiting examples of linker sequences are discussed further herein and include, but are not limited to, GS linker sequences of various lengths. When the heavy chain-only variable region is in a bivalent configuration, each of the two heavy chain-only variable region domains can have binding affinity for the same antigen or different antigens (e.g., for different epitopes on the same protein, for two different proteins, etc.). However, unless specifically stated otherwise, a heavy chain-only variable region designated as being in a "bivalent configuration" is understood to comprise two identical heavy chain-only variable region domains connected by a linker sequence, each of which has binding affinity for the same target antigen.

[0059] Aspects of the present disclosure include antibodies with multispecific configurations, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations are known and used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0060] Various methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 25) (n=1) and GGGGSGGGGS (SEQ ID NO: 26) (n=2). Other suitable linkers can also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0061] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domains are derived from or share sequence identity with the variable regions of the antibody heavy or light chains. Portions of such variable regions are V H and / or V L Gene segments, D and J H Gene segment, or J L The variable region may be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It may be encoded by a gene segment.

[0062] The TCA binding compounds utilize "heavy chain-only antibodies" or "heavy chain antibodies" or "heavy chain polypeptides," which, as used herein, refer to single-chain antibodies comprising heavy chain constant regions CH2 and / or CH3 and / or CH4, but not CH1 domains. In one embodiment, a heavy chain-only antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and CH2 domain. In a further embodiment, a heavy chain antibody is comprised of an antigen-binding domain, at least a portion of a hinge region, and CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domains have been truncated are also included herein. In a further embodiment, the heavy chain is comprised of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but not the hinge region. Heavy-chain-only antibodies can be in the form of a dimer in which two heavy chains are disulfide-bonded or otherwise covalently or non-covalently bound to each other, and can optionally include an asymmetric interface between one or more of the CH domains to facilitate proper pairing between the polypeptide chains. Heavy-chain antibodies may belong to the IgG subclass, although antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, heavy-chain antibodies are of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 or IgG4 subtype. Non-limiting examples of TCA-binding compounds are described, for example, in International Publication Nos. WO 2017 / 223111 and WO 2018 / 052503, the disclosures of which are incorporated herein by reference in their entireties.

[0063] Heavy chain antibodies constitute approximately one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding portion is called a VHH domain, which represents the smallest naturally occurring intact antigen-binding site, only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy-chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, which is called VNAR. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003), Nuttall et al.Function and Bioinformatics 55,187-197(2004), Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0064] As used herein, the terms "CD19" and "cluster of differentiation 19" refer to a molecule expressed during all phases of B cell development up to terminal differentiation into plasma cells. The term "CD19" includes the CD19 protein of any human and non-human animal species, and specifically includes human CD19 as well as CD19 of non-human mammals.

[0065] As used herein, the term "human CD19" includes any variant, isoform, and species homolog of human CD19 (UniProt P15391), regardless of its source or mode of preparation. Thus, "human CD19" includes human CD19 naturally expressed by cells and CD19 expressed on cells transfected with the human CD19 gene.

[0066] The term "CD3" refers to the human CD3 protein multisubunit complex. The CD3 protein multisubunit complex is composed of six distinct polypeptide chains. These include the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), two CD3 epsilon chains (SwissProt P07766), and one CD3 zeta chain homodimer (SwissProt 20963), which associate with the T cell receptor alpha and beta chains. Unless otherwise specified, the term "CD3" includes any CD3 variants, isoforms, and species homologs that are naturally expressed by cells (including T cells) or that can be expressed on cells transfected with genes or cDNAs encoding those polypeptides.

[0067] A "CD19xCD3 antibody" is a multispecific heavy chain-only antibody, such as a bispecific heavy chain-only antibody, that contains two different antigen-binding regions, one of which specifically binds to CD19 and the other of which specifically binds to CD3.

[0068] The terms "anti-CD19 heavy chain-only antibody," "CD19 heavy chain-only antibody," "anti-CD19 heavy chain antibody," and "CD19 heavy chain antibody" are used interchangeably herein and refer to heavy chain-only antibodies, as defined herein above, that immunospecifically bind to CD19, including human CD19, as defined herein above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice, that express human immunoglobulins, including UniRats™, which produce the human anti-CD19 UniAb™ antibody, as defined herein above.

[0069] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0070] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0071] Antibodies of the present disclosure include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinities, e.g., higher-order polyepitopic specificities, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody," "multispecific heavy chain-only antibody," "multispecific heavy chain antibody," "multispecific UniAb™," and "multispecific binding compound" are used in the broadest sense herein to encompass all antibodies with more than one binding specificity. Multispecific heavy chain anti-CD19 antibodies of the present disclosure specifically include antibodies (i.e., bivalent and monoparatopic) that immunospecifically bind to one single epitope on the CD19 protein, such as human CD19, and to an epitope on a different protein, e.g., the CD3 protein. Multispecific heavy chain anti-CD19 antibodies of the present disclosure specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on a CD19 protein, such as human CD19 (i.e., bivalent and biparatopic). Multispecific heavy chain anti-CD19 antibodies of the present disclosure also specifically include antibodies that immunospecifically bind to an epitope on a CD19 protein, such as human CD19, and an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 (i.e., bivalent and biparatopic). Multispecific heavy chain anti-CD19 antibodies of the present disclosure also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on a CD19 protein, such as a human CD19 protein, and an epitope on a different protein, such as, for example, a CD3 protein, such as human CD3 protein (i.e., trivalent and biparatopic).

[0072] Antibodies of the present disclosure include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity as well as antibodies comprising more than one binding unit having the same binding specificity. The terms "monospecific antibody," "monospecific heavy chain-only antibody," "monospecific heavy chain antibody," and "monospecific UniAb™" are used in the broadest sense herein to encompass all antibodies having one binding specificity. Monospecific heavy chain anti-CD19 antibodies of the present disclosure specifically include antibodies (monovalent and monospecific) that immunospecifically bind to one epitope on a CD19 protein, such as human CD19. Monospecific heavy chain anti-CD19 antibodies of the present disclosure also specifically include antibodies with more than one binding unit (e.g., multivalent antibodies) that immunospecifically bind to an epitope on a CD19 protein, such as human CD19. For example, a monospecific antibody according to an embodiment of the present disclosure can comprise a heavy chain variable region comprising two antigen-binding domains, each of which binds to the same epitope on the CD19 protein (i.e., bivalent and monospecific).

[0073] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and will react with many different antibodies. The term specifically includes continuous and conformational epitopes.

[0074] "Epitope mapping" is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes can be continuous or structural epitopes. Continuous epitopes are formed by a contiguous sequence of amino acids in a protein. Structural epitopes are formed from amino acids that are discontinuous in the protein sequence but are brought together when the protein folds into its three-dimensional structure.

[0075] The term "valency" as used herein refers to a specific number of binding sites in an antibody molecule.

[0076] A "monovalent" antibody has one binding site. Thus, monovalent antibodies are also monospecific.

[0077] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific antibodies according to the present disclosure are at least bivalent, and may be trivalent, tetravalent, or otherwise multivalent. Bivalent antibodies according to aspects of the present disclosure may have two binding sites for the same epitope (i.e., bivalent, monoparatopic) or two different epitopes (i.e., bivalent, biparatopic).

[0078] A wide variety of methods and protein conformations are known and used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0079] The term "tri-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain, and an antigen-binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domain is derived from or shares sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions are V H and / or V L Gene segments, D and J H Gene segment, or J L The variable region may be encoded by a rearranged VH DJ H , V L DJ H , V H J L , or V L J L The TCA protein may be encoded by a gene segment. The TCA protein utilizes a heavy chain-only antibody as defined above.

[0080] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation. The term "human antigen" specifically includes heavy chain-only antigens with human heavy chain variable region sequences produced by transgenic animals such as transgenic rats or mice, particularly UniAbs™ produced by UniRats™ as defined above.

[0081] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with a non-human or artificial Fc region and a human idiotype. Such immunoglobulins can be isolated from animals of the present disclosure that have been engineered to produce such chimeric antibodies.

[0082] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcRs, are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0083] "Human effector cells" are leukocytes that express a receptor, such as a T cell receptor or FcR, and perform effector function. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells may be isolated from their native source, for example, from blood or PBMCs as described herein.

[0084] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0085] Antibody "effector functions" refer to biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

[0086] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0087] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0088] "B-cell Hodgkin lymphoma (B-NHL) is defined to include diffuse large B-cell lymphoma (DLBCL; including subtypes such as primary mediastinal B-cell lymphoma), high-grade B-cell lymphoma (HGBL; including subtypes such as HGBL with MYC and BCL2 and / or BCL6 rearrangements), transformed indolent NHL including Richter's transformed NHL, mantle cell lymphoma (MCL), follicular lymphoma (FL, grades 1-3), or marginal zone lymphoma (MZL).

[0089] "CD19 positive" is or refers to expression of CD19 in 50% or more of the tumor cells as assessed by a pathologist using immunohistochemistry (IHC) or flow cytometry.

[0090] The term "characterized by expression of CD19" refers broadly to any disease or disorder in which CD19 expression is associated with or contributes to one or more pathological processes characteristic of the disease or disorder, including, but not limited to, B-cell neoplasms.

[0091] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens more quickly and tend to remain bound to them.

[0092] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined by BioLayer Interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor is loaded with a mouse-Fc fusion antigen and then immersed in a well containing an antibody to measure the concentration-dependent association rate (k). The antibody dissociation rate (koff) is measured in the final step, where the sensor is immersed in a well containing only buffer. Kd is the ratio of koff / koff. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0093] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., causing regression of the disease. A therapeutic agent may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is particularly meaningful if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment desirably occurs before complete loss of function in the affected tissue. The therapy of interest may be administered during, and in some cases after, the symptomatic stage of the disease.

[0094] By "therapeutically effective amount" is intended the amount of active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, improves, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

[0095] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or treated. In certain aspects, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, and the like. Subjects may be humans, but also include other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like.

[0096] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0097] A "sterile" formulation is aseptic or free or essentially free of all living microorganisms and their spores. A "frozen" formulation is one at a temperature below 0°C.

[0098] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991), and Jones, A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature for a selected time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection), assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis, amino- or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis to compare reduced and intact antibodies, peptide map (e.g., trypsin or LYS-C) analysis, assessing antibody biological activity or antigen-binding function, etc. Instability can involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differences in glycosylation, etc.

[0099] Abbreviations used herein include the following: ADA (anti-drug antibody), ADT (androgen deprivation therapy), AE (adverse event), ALT (alanine aminotransferase), ANC (absolute neutrophil count), AR (androgen receptor), AST (aspartate aminotransferase), AUC (area under the concentration-time curve), AUCt (area under the serum concentration-time curve from time zero to the time of the last measurable concentration), CAR (chimeric antigen receptor), CARTOX (CAR-T cell therapy-related toxicity), CBR (clinical benefit rate), CI (confidence interval), CL (clearance), Cmax (maximum observed serum concentration), CNS (central nervous system), CR (complete response), CRS (cytokine release syndrome), Css, trough (trough concentration at steady state), CT (computed tomography), CTCAE (Common Terminology Criteria for Adverse Events), DLT (dose-limiting toxicity), DOR (duration of response), ECG (electrocardiogram), ECOG (Eastern Cooperative Oncology Group), eCRF (electronic case report form), EDC (electronic data capture), eGFR (estimated glomerular filtration rate), EOI (end of infusion), EOT (end of treatment), FIH (first-in-human), FISH (fluorescence in situ hybridization), FL (follicular lymphoma), GCP (good clinical practice guidelines), G-CSF (granulocyte colony-stimulating factor), GLP (good laboratory practice guidelines for non-clinical studies on drug safety), HBsAg (hepatitis B surface antigen), HBV (hepatitis B virus), HCV (hepatitis C virus), HCV Ab (Hepatitis C virus antibody), HIV (Human immunodeficiency virus), IB (Investigator Brochure), ICE (Immune Effector Cell Encephalopathy), ICF (Informed Consent Form), ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use), IEC (Independent Ethics Committee), IMT (Immune-Mediated Toxicity), IRB (Institutional Review Board), IV (Intravenous), mAb (Monoclonal Antibody), MABEL (Minimum Estimated Effective Level), MED (Minimum Effective Dose), MedDRA (Medical Regulatory Terminology), MR (Minimum Response), MRI (Magnetic Resonance Imaging), MTD (Maximum Tolerated Dose), NA (Not Applicable), NCA (Non-compartmental Analysis);NCCN (National Comprehensive Cancer Network), NCI (National Cancer Institute), B-NHL (B-cell non-Hodgkin's lymphoma), NLCB (no longer clinically beneficial), NT (neurotoxicity), ORR (objective response rate), OS (overall survival), PARPi (poly-ADP-ribose-polymerase inhibitor), PBMC (peripheral blood mononuclear cells), PC (positive control), PD (pharmacodynamics or disease progression), PET (positron emission tomography), PI (prescribing information), PK (pharmacokinetics), PFS (progression-free survival), PO (per os, oral), PR (partial response), PT (prothrombin time), Q3W (once every 3 weeks), QTc (QT interval corrected for heart rate), RLT (radioligand therapy), rPFS (radiographic progression-free survival), RP2D (recommended phase 2 dose), RR (relapsed or refractory), SAE (serious adverse event), SCT (stem cell transplant), SD (stable disease), SMG (safety monitoring group), SUSAR (suspected unexpected serious adverse reaction), T; 1 / 2 or t 1 / 2 (terminal elimination half-life), T-BsAb (T cell-engaging bispecific antibody), TEAE (treatment-emergent adverse event), TLS (tumor lysis syndrome), Tmax (time to maximum observed serum concentration), TTP (time to disease progression), TTR (time to response), ULN (upper limit of normal), US (United States), V1 (central compartment volume), Vss (volume of distribution at steady state), WHO (World Health Organization).

[0100] II.TNB-486 (anti-CD3 / anti-CD19) The present disclosure relates to methods of treating non-Hodgkin's lymphoma by administering a bispecific triple-chain antibody-like molecule (TCA) to a patient in need thereof. In some embodiments, the TCA is designated TNB-486 and comprises an anti-CD3 binding domain paired with an anti-CD19 VH binding domain as shown in FIG. 4 , wherein the anti-CD3 VH domain and the anti-CD3 VL domain together have binding affinity for CD3, and the TCA further comprises a heavy chain variable domain of a heavy chain-only antibody in a monovalent configuration and has binding affinity for CD19, and the TCA further comprises a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence comprising S228P, F234A, L235A, and T366W mutations (knob) and a second heavy chain constant region sequence comprising S228P, F234A, L235A, T366S, L368A, and Y407V mutations (hole).

[0101] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3, in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 6, in a human VL framework. The CD3-binding VH domain and the light chain variable domain together have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 7. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 7. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 8. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 8.

[0102] Multispecific antibodies comprising the above-described CD3-binding VH domain and light chain variable domain have advantageous properties, as described, for example, in PCT Publication No. WO2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety.

[0103] [Table 1]

[0104] [Table 2]

[0105] [Table 3]

[0106] [Table 4-1]

[0107] [Table 4-2]

[0108] [Table 4-3]

[0109] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, but not limited to, bispecific three-chain antibody-like molecules. In some embodiments, a bispecific antibody may comprise at least one heavy chain variable region that has binding specificity for CD19 and at least one heavy chain variable region that has binding specificity for a different protein, e.g., CD3. In some embodiments, a bispecific antibody may comprise a heavy chain / light chain pair that has binding specificity for a first antigen, a heavy chain derived from a heavy-chain-only antibody that comprises an Fc portion that comprises a CH2 and / or CH3 and / or CH4 domain in the absence of a CH1 domain, and an antigen-binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, in a monovalent or bivalent configuration. In one particular embodiment, the bispecific antibody comprises a heavy chain / light chain pair that has binding specificity for an antigen on an effector cell (e.g., a CD3 protein on a T cell) and a heavy chain from a heavy chain-only antibody that comprises an antigen-binding domain that has binding specificity for CD19, in a monovalent or bivalent configuration.

[0110] In some embodiments, when the antibody of the present disclosure is a bispecific antibody, one arm of the antibody (one binding moiety, or one binding unit) may be specific for human CD19, and the other arm may be specific for a target cell, a tumor-associated antigen, a target antigen such as an integrin, a pathogen antigen, a checkpoint protein, etc. Target cells specifically include cancer cells. In some embodiments, one arm of the antibody (one binding moiety, or one binding unit) is specific for human CD19, and the other arm is specific for CD3.

[0111] In one preferred embodiment, the antibody is a bispecific TCA comprising a first polypeptide comprising SEQ ID NO: 11, a second polypeptide comprising SEQ ID NO: 18, and a third polypeptide comprising SEQ ID NO: 20. In some embodiments, CDR1 comprises a polypeptide comprising SEQ ID NO: 21. In some embodiments, CDR2 comprises a polypeptide comprising SEQ ID NO: 22. In some embodiments, CDR3 comprises a polypeptide comprising SEQ ID NO: 23. This antibody, also referred to as TNB-486, is also described in U.S. Pat. No. 11,390,681(B2) and PCT Publication No. WO2021 / 222578, the disclosures of each of which are incorporated herein by reference in their entireties.

[0112] III. Antibody Preparation The multispecific antibodies of the present disclosure can be prepared by methods known in the art. In a preferred embodiment, the heavy chain antibodies of the present disclosure are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which endogenous immunoglobulin genes have been knocked out or disabled. In a preferred embodiment, the heavy chain antibodies of the present disclosure are produced in UniRat™ mice. UniRat™ mice have their endogenous immunoglobulin genes silenced and use a human immunoglobulin heavy chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs. While endogenous immunoglobulin loci in rats can be knocked out or silenced using various techniques, in UniRat™, the endogenous rat heavy chain J locus, light chain Cκ locus, and light chain Cλ locus were inactivated using zinc finger (endo)nuclease (ZNF) technology. ZNF constructs for microinjection into oocytes can produce IgH and IgL knockout (KO) strains. For details, see, e.g., Geurts et al., 2009, Science 325:433. Characterization of Ig heavy chain knockout rats was reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. An advantage of ZNF technology is that non-homologous end-joining to silence genes or loci via deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced in UniRat™ are called UniAbs™ and can bind epitopes that cannot be targeted by conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and multi-specific applications.

[0113] In addition to UniAbs™, specifically included herein are camelid VHH frameworks and mutations, as well as heavy chain-only antibodies lacking their functional VH regions. Such heavy chain-only antibodies can be produced, for example, in transgenic rats or mice containing a fully human heavy chain-only locus as described in WO 2006 / 008548, although other transgenic mammals such as rabbits, guinea pigs, and rats can also be used, with rats and mice being preferred. Heavy chain-only antibodies, including their VHH or VH functional fragments, can also be produced by recombinant DNA technology, for example, by expressing encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0114] Heavy chain-only antibody domains combine the advantages of antibodies and small molecule drugs, can be monovalent or multivalent, have low toxicity, and are cost-effective to produce. Due to their small size, these domains are easy to administer, including orally or topically, and are characterized by high stability, including gastrointestinal stability, and their half-life can be tailored to the desired use or indication. In addition, VH and VHH domains of HCAbs can be produced in a cost-effective manner.

[0115] In certain embodiments, heavy chain antibodies of the present disclosure, including UniAbs™, have a replacement of the native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) with another amino acid residue capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position. Such a hydrophobic patch is normally buried at the interface with the antibody light chain constant region, but in HCAbs it is surface-exposed and is responsible, at least in part, for undesired aggregation and light chain association of HCAbs. The substituted amino acid residue is preferably charged, more preferably positively charged, such as lysine (Lys, K), arginine (Arg, R), or histidine (His, H), preferably arginine (R). In a preferred embodiment, heavy chain-only antibodies derived from transgenic animals contain a Trp to Arg mutation at position 101. The resulting HCAbs preferably have high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0116] As part of the present invention, human IgG anti-CD19 heavy chain antibodies with unique sequences derived from UniRat™ animals (UniAb™) were identified that bind to human CD19 in ELISA (recombinant CD19 extracellular domain) protein and cell binding assays. The identified heavy chain variable region (VH) sequences (see Figure 2) are positive for human CD19 protein binding and / or binding to CD19+ cells, and are all negative for binding to cells that do not express CD19.

[0117] The antibodies described herein bind the CD19-positive Burkitt's lymphoma cell lines Daudi (ATCC® CCL-213™), Raji (ATCC® CCL-86™), and Ramos (ATCC® CRL-1596™), and some are cross-reactive with the CD19 protein of cynomolgus monkeys. In addition, they can be engineered to provide cross-reactivity with the CD19 protein of any animal species, if desired.

[0118] Anti-CD19 heavy chain antibodies, such as the UniAbs™ herein, are administered in a concentration of about 10 -6 ~about 10 -11 For example, but not limited to, about 10 -6 ~about 10 -10 Around 10 -6 ~about 10 -9 Around 10 -6 ~about 10 -8 Around 10 -8 ~about 10 -11 Around 10 -8 ~about 10 -10 Around 10 -8 ~about 10 -9 Around 10 -9 ~about 10 -11 Around 10 -9 ~about 10 -10 The antibodies may have an affinity for CD19 with a Kd around, or any value within these ranges. Affinity selection may be confirmed using biological evaluations to modulate, e.g., block, CD19 biological activity, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0119] Heavy chain antibodies that bind to non-overlapping epitopes on the CD19 protein, such as UniAbs™, can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between a known antibody that binds to a target antigen and an antibody of interest can be used. Using this approach, a set of antibodies can be divided into antibodies that compete with the reference antibody and those that do not compete. Non-competing antibodies are identified as those that bind to distinct epitopes that do not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind the captured antigen. This can be done using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR Imager (Ibis technologies BV), as well as on biolayer interferometry platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the Examples herein.

[0120] Typically, an antibody "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to a target antigen, as determined by standard techniques, such as the competitive binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more.

[0121] Pharmaceutical Composition Another aspect of the present disclosure is to provide pharmaceutical compositions comprising one or more multispecific binding compounds of the present disclosure in admixture with a suitable pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carriers include, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0122] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to CD19. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificity for two or more non-overlapping epitopes on the CD19 protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) that has binding specificity for CD19 and has binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, such as the CD3 protein on a T cell).

[0123] Pharmaceutical compositions of antibodies used in accordance with the present disclosure are prepared for storage by mixing the protein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), e.g., in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0124] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dosage for a single administration). The formulation will depend on the route of administration chosen. The antibodies herein can be administered by intravenous injection or infusion, or subcutaneously. For injectable administration, the antibodies herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can include a carrier, excipient, or stabilizer as discussed above. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0125] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for heavy chain antibodies, including the UniAbs™ of the present disclosure. Subcutaneous antibody formulations are described, for example, in U.S. Patent Application Publication Nos. 20160355591 and 20160166689.

[0126] IV.How to use The antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of a target protein (e.g., CD3, CD19), including, but not limited to, the conditions and diseases further described herein. In a preferred aspect, the antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of CD19.

[0127] The pharmaceutical compositions herein comprising anti-CD19 antibodies can be used to treat disorders characterized by expression of CD19, including, but not limited to, non-Hodgkin's lymphoma.

[0128] The effective dose of the compositions of the present disclosure for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, e.g., companion animals such as dogs, cats, horses, and laboratory mammals such as rabbits, mice, rats, and the like, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0129] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of a solid (e.g., lyophilized) composition. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, or copolymers, to enhance adjuvant effect, as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present disclosure can be administered in the form of depot injection or implant preparations, which can be formulated to allow sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all US Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0130] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosage can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0131] Compositions for administration will generally contain an antibody or other agent (e.g., another ablative agent) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, in accordance with the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0132] Kits containing the disclosed active agents and formulations thereof and instructions for use are also within the scope of this disclosure. The kits may further include at least one additional reagent, such as a chemotherapeutic agent. The kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any written or recorded material supplied on or with the kit, or otherwise accompanying the kit.

[0133] Embodiments of the present invention include methods for evaluating the safety, clinical pharmacokinetics (PK), and clinical activity of TNB-486 in subjects with B-NHL. As described above, B-NHL subtypes may include, but are not limited to, chronic lymphocytic leukemia / small lymphocytic lymphoma, follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL), and mantle cell lymphoma (MCL). For the purposes of this study, B-NHL subtypes include follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL), and mantle cell lymphoma (MCL).

[0134] In some embodiments, the patient has received at least two lines of systemic therapy approved for B-NHL. In some embodiments, the patient has previously been exposed to, is intolerant of, is ineligible for, or has declined typical second-line therapy, such as high-dose chemotherapy followed by autologous (stem cell transplant (SCT)).

[0135] Embodiments of the present disclosure include methods involving monotherapy dose escalation (Part 1, Arm A) and monotherapy dose expansion (Part 2, Arms B and C). A schematic diagram of the study design for this administration is shown in Figure 1A. Some embodiments include methods involving TNB-486 dose escalation in combination with R-CHOP (Part 3, Arm D). A schematic diagram of the administration of the combination dosing regimen is shown in Figure 1B.

[0136] Embodiments of the present disclosure include administration methods in which dose-limiting toxicity (DLT) occurs at a lower rate than other administration methods. In some embodiments, the DLT that occurs is cytokine release syndrome (CRS). CRS and its severity grades are described herein. In some embodiments, the patient does not experience CRS or only experiences Grade 1 CRS. In some embodiments, the patient does not experience CRS. In some embodiments, the patient does not experience CRS or only experiences Grade 1 or Grade 2 CRS. In some embodiments, any CRS experienced by the patient resolves without CRS treatment. In some embodiments, any CRS experienced by the patient resolves with CRS treatment. In embodiments, CRS is treated by administration of tocilizumab.

[0137] In some embodiments of the methods, the patient has less than a 20% chance of experiencing Grade 2 or Grade 3 CRS. In embodiments, the patient has less than a 24% chance of experiencing Grade 2 or Grade 3 CRS. In embodiments, the patient has a 40% chance of not experiencing CRS.

[0138] In embodiments, the patient experiences CRS only during the first administration cycle. In embodiments, the patient has about a 53% or less chance of experiencing CRS on day 1 of the first administration cycle. In embodiments, the patient has about a 15% or less chance of experiencing CRS on day 15 of the first administration cycle. In embodiments, the patient does not experience CRS during the second administration cycle. In embodiments, the patient does not experience CRS during the second administration cycle or any subsequent administration cycle.

[0139] Monotherapy Dose Escalation (Part 1, Arm A): In some embodiments, the methods involve evaluating the safety, tolerability, PK, and PD profile of single-agent TNB-486 therapy administered once every four weeks (Q2W, 28-day cycles) in patients with B-NHL who have received at least two prior lines of therapy. In some embodiments, patients have previously been exposed to, are intolerant of, are ineligible for, or have declined typical second-line therapy, such as high-dose chemotherapy followed by autologous stem cell transplant (SCT). In Arm A, the cohort initially enrolls a single subject.

[0140] In some embodiments, the method involves administering a single dose of TNB-486 in a 28-day cycle for at least one cycle. In some embodiments, the TNB-486 dose is selected from the group consisting of 30 μg, 90 μg, 270 μg, 800 μg, 2400 μg, 7200 μg, 15000 μg, and 30000 μg. In some embodiments, the method involves modifying the dosing frequency to include additional time between doses. In some embodiments, the treatment cycle is repeated two or more times. In some embodiments, TNB-486 is administered to the patient as monotherapy. In some embodiments, TNB-486 is administered by intravenous infusion (IV).

[0141] In some embodiments, the patient will receive one or more priming doses prior to the target dose. In some embodiments, the patient will receive a single priming dose on day 1 of the first administration cycle. In some embodiments, the patient will receive two priming doses. In some embodiments, the patient will receive two priming doses, with the first priming dose administered on day 1 of the first administration cycle and the second priming dose administered on day 2 of the first administration cycle.

[0142] In some embodiments, Arm A involves a dose-escalation design to evaluate the safety, tolerability, PK, and PD profiles of single-agent TNB-486 administered Q2W in up to 24 B-NHL patients who have received at least two prior lines of therapy. In some embodiments, patients have previously been exposed to, are intolerant of, are ineligible for, have declined therapy for, or are not suitable candidates for, a treatment regimen known to provide clinical benefit in B-NHL. In Arm A, a cohort initially enrolls a single subject. In some embodiments, nine dose levels of TNB-486 are evaluated (Table), although the number of dose levels tested will depend on safety, PK / PD, and activity data.

[0143] In Arm A, the cohort will initially enroll a single subject (Figure 2 and Table 5). Eight dose levels are proposed for TNB-486 (Table 5), although the number of dose levels tested will depend on tolerability. In some embodiments, dose levels are selected from the following: 30 μg, 90 μg, 270 μg, 800 μg, 2400 μg, 7200 μg, 15000 μg, 30000 μg, 40000 μg, 50000 μg, 60000 μg, 70000 μg, 80000 μg, 90000 μg, and 100000 μg.

[0144] In embodiments where at least one priming dose is administered, the priming dose is 150 μg to 1500 μg. In embodiments, the priming dose is 175 μg to 1250 μg. In embodiments, the priming dose is 200 μg to 1000 μg. In embodiments, the priming dose is from In embodiments, the priming dose is from 200 μg to 500 μg. In embodiments, the priming dose is from 250 μg to 1000 μg. In embodiments, the priming dose is from In embodiments, the priming dose is from 250 μg to 500 μg. In embodiments, the priming dose is from In embodiments, the priming dose is from 270 μg to 1000 μg.

[0145] In embodiments of the method, two priming doses are administered. In embodiments where two priming doses are administered, the first priming dose is between about 150 μg and about 540 μg. In embodiments, the first priming dose is between about 175 μg and about 500 μg. In embodiments, the first priming dose is between about 200 μg and about 400 μg. In embodiments, the first priming dose is between about 200 μg and about 300 μg. In embodiments, the first priming dose is between about 250 μg and about 300 μg. In embodiments, the first priming dose is about 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, or 350 μg. In embodiments, the first priming dose is about 270 μg. In embodiments, the first priming dose is 270 μg.

[0146] In embodiments where two priming doses are administered, the second priming dose is about 800 μg to about 1200 μg. In embodiments, the second priming dose is about 850 μg to about 1150 μg. In embodiments, the second priming dose is about 900 μg to about 1100 μg. In embodiments, the second priming dose is about 950 μg to about 1050 μg. In embodiments, the second priming dose is about 950 μg, 960 μg, 970 μg, 980 μg, 990 μg, 1000 μg, 1010 μg, 1020 μg, 1030 μg, 1040 μg, or 1050 μg. In embodiments, the second priming dose is about 1000 μg. In embodiments, the second priming dose is 1000 μg.

[0147] In some embodiments, two priming doses are administered, the first priming dose being about 270 μg and the second priming dose being about 1000 μg.

[0148] [Table 5] Abbreviations: DLT = dose-limiting toxicity; MTD = maximum tolerated dose; N = cohort number; RP2D = recommended phase 2 dose; SMG = safety monitoring group. aDose tapering from any dose level (except starting at 30 μg) may be performed to refine the determination of the MTD and / or RP2D. b A single priming dose will be explored in cohorts 4b, 5b, 6a, and 7a. Starting with cohort 4b, cohorts Na, b, c, etc. may be run in parallel. Not all dose level cohorts will be enrolled based on new safety data. C Cohort 6c is closed to enrollment d The approximate number of subjects is based on the lack of dose-related toxicity in any cohort. The actual number of subjects will depend on safety and other findings. No more than three subjects will be enrolled simultaneously in a given subcohort. e Cohorts 5d, 6d, and 7d are not part of the dose escalation design but are considered backfills for cohorts 5b, 6a, and 7a, respectively. No formal DLT assessments are planned for the cohorts considered backfills. Once cohort 7a is deemed safe for SMG escalation as guided by BOIN, enrollment into cohort 8d may begin.

[0149] Upon the first occurrence of a grade 2 or higher adverse event (AE) not clearly attributable to the subject's underlying malignancy or other unrelated cause, the corresponding cohort and all subsequent cohorts in Arm A will be expanded to three subjects and will follow a standard 3+3 dose escalation design. If a DLT occurs in a single subject cohort, the cohort will be expanded to six subjects. If a second DLT occurs in a cohort, the previous dose is the MTD. If only one DLT is observed, subsequent cohorts will progress according to a 3+3 dose escalation design.

[0150] If a response (PR or CR) is observed in a single subject cohort, subsequently enrolled cohorts will progress according to a 3+3 dose escalation design.

[0151] [Table 6] Abbreviations: DLT = dose-limiting toxicity; MTD = maximum tolerated dose.

[0152] In some embodiments, dose escalation begins with a Q2W dosing schedule, which may be changed after cumulative review of safety and PK data and protocol amendments. Administration of the first dose of TNB-486 to the first subject in each cohort must await completion of the first cycle (4 weeks) of the previous dose level and review of safety data by the medical monitor (Syneos, Inc. and TeneoTwo) and the principal investigator (or designated secondary investigator, hereafter referred to as SMG). Input from biostatistics, PK, and other experts will be sought as needed.

[0153] In some embodiments, cohorts receiving intermediate doses between the doses proposed above may be conducted based on ongoing review of the SMG for new safety and clinical pharmacology data. Additionally, it may be necessary to switch to an alternative dosing regimen. For example, dosing may be switched to a different frequency (e.g., once every three weeks), or some cycles may be consistently eliminated from the dosing schedule (e.g., every scheduled third cycle may be stopped). When switching the dosing schedule to occur more frequently, the dose change must not result in a predicted steady-state concentration (CSS) or Cmax greater than that specified for the next lower dose level. When evaluating new intermediate dose levels and / or new dosing schedules, a protocol amendment with appropriate justification will be submitted.

[0154] Dose escalation and selection of MTD / RP2D dose: In some embodiments, DLT criteria are used to make decisions regarding dose escalation. Dose escalation decisions are made by the SMG based on clinically significant toxicity, DLT events, and PK and PD findings (if available). A simple majority of the SMG is required to proceed with dose escalation.

[0155] Dose escalation will proceed as follows (and as shown in Figure 2): In single-subject cohorts, if the first DLT-evaluable subject in a given cohort completes safety evaluation during the first cycle without evidence of activity and without experiencing a Grade ≥ 2 AE not clearly attributable to their underlying malignancy or other unrelated cause, proceed with escalation to the next proposed dose level. If a subject in a single subject cohort experiences a non-DLT Grade ≥ 2 AE not clearly attributable to their underlying malignancy or other unrelated cause, expand that same dose level to three subjects. If the first three DLT-evaluable subjects in a cohort of three subjects complete the safety assessment during the first cycle without experiencing a DLT, proceed with escalation to the next proposed dose level. If one subject at a dose level experiences a DLT (including a single-subject cohort), expand that same dose level to six subjects. If 1 of 6 evaluable subjects experiences a DLT, proceed with escalation to the next proposed dose level. If 2 of 6 subjects experience a DLT, dose escalation will be stopped because the MTD has been exceeded.

[0156] In some embodiments, if the SMG safety review of Cycle 1 for Cohort N is completed and the dose is deemed safe (e.g., the SMG approves further dose escalation or determines that the Cohort N dose is the RP2D), any subjects remaining on the study at the lower dose of TNB-486 may subsequently be treated at the dose assigned to Cohort N (e.g., if the SMG review for Cohort 5 is completed and a decision is made to escalate to Cohort 6, any subjects still on the study from Cohorts 1-4 may have their dose increased to the dose corresponding to Cohort 5). Subject eligibility for such escalation must be approved by the SMG and is determined on a case-by-case basis. At a minimum, to be eligible, a subject must have previously received at least two cycles of TNB-486 at the subject's current dose, have been free of any drug-related toxicity that resulted in a dose reduction, and have had at least one post-dose disease assessment. Subjects must have stable disease or better and must not have experienced any Grade 2 or higher AEs on their most recent dose of TNB-486 that are not clearly attributable to the underlying disease or other unrelated cause.

[0157] In some embodiments, if the data available during SMG review of Cohort N are unclear regarding further escalation (e.g., based on non-DLT AEs, plateau efficacy, occurrence of questionable RP2D), Cohort N and / or Cohort N-1 may be expanded to a maximum of 9 subjects each, at the discretion of the SMG.

[0158] Medication preparation: Based on the nonclinical pharmacology of TNB-486, a lower CRS rate is expected than with other T cell-engaging bispecific antibodies. However, based on TNB-486's mechanism of action, the most likely DLT is still grade 3 or higher CRS. Cytokine release syndrome will be managed at the discretion of the principal investigator and according to institutional guidelines (if institutional guidelines have not been established, a guide for the management of CRS is provided in Figure 3).

[0159] In some embodiments, if a Grade 3 or higher CRS event occurs in Cohort N during the DLT period, or at the discretion of the SMG, doses equal to or lower than the Cohort N dose will be designated as "priming doses" and dose administration in subsequent cohorts (Cohort N+1 and beyond) will be modified as follows: On Day 1 of Cycle 1, subjects will receive a priming dose. The priming dose in Cohorts N+1 and above will not exceed the dose administered in Cohort N. In embodiments in which a second priming dose is administered, on Day 8 of Cycle 1, the subject receives the second priming dose. Beginning on Day 15 of Cycle 1, subjects will receive the full dose corresponding to their enrolled cohort. In Cohort N+1, the full dose will not exceed the reserve dose (i.e., the dose administered to Cohort N) by more than 50%. The full dose administered in cohorts N+2 and above must not exceed +100% of the full dose in the immediately preceding cohort. If two Grade ≥ 3 CRS AEs occur in the same cohort, the full dose escalation should not subsequently exceed +50% of the immediately preceding cohort. If a DLT occurs, dose escalation to full dose should not subsequently exceed +33% of the immediately preceding cohort.

[0160] If, after dosing on Day 1 of Cycle 1, the subject experiences any Grade 3 or higher toxicity not clearly attributable to the subject's underlying malignancy or other unrelated cause, administration of the full dose on Day 15 of Cycle 1 must await approval by the medical monitor. At a minimum, CRS must resolve to Grade 1 or less before the full dose is administered. The dose and timing of the full dose for such subjects may be modified at the discretion of the medical monitor and investigator. If a subject has their full dose reduced in Cycle 1, they are considered non-DLT evaluable and should be replaced unless they experience a DLT. In subsequent cycles, subjects will receive the full dose on Days 1 and 15.

[0161] If administration of the full dose is delayed in Cycle 1 after administration of the priming dose, the DLT period for that subject should be extended to the same extent (e.g., if the full dose is delayed by 4 days, the DLT period will also be extended by 4 days). For clarity, treatment-emergent adverse events (TEAEs) that meet the criteria for a DLT will be treated as DLTs regardless of whether they occur after administration of the priming dose or the full dose.

[0162] Monotherapy Dose Expansion (Part 2, Arm B): In some embodiments, the method involves assessing the MTD (or RP2D) of TNB-486 monotherapy in approximately 30 subjects with biopsy-proven RR DLBCL or HGBL. To be eligible for this study, subjects must have received two or more prior lines of therapy and must not be suitable candidates for treatment regimens known to provide clinical benefit in DLBCL / HGBL. At least 15 subjects must not have been previously treated with CD19-targeted therapy. After selecting the MTD (or RP2D) based on data from the monotherapy dose escalation (Part 1, Arm A), Arm B will be initiated. The MTD (or RP2D) and dosing frequency for Arm B will be selected by the SMG based on safety, tolerability, and PK / PD data collected during the dose escalation portion of the study.

[0163] Monotherapy Dose Expansion (Part 2, Arm C): In some embodiments, the method involves assessing the MTD (or RP2D) of TNB-486 monotherapy in approximately 20 subjects with biopsy-proven RR FL (grade 1-3a). To be eligible for this study, subjects must have received two or more prior lines of therapy and must not be suitable candidates for treatment regimens known to provide clinical benefit in FL. At least 10 subjects must not have been previously treated with CD19-targeted therapy. After selecting the MTD (or RP2D) based on data from the monotherapy dose escalation (Part 1, Arm A), Arm C will be initiated. The MTD (or RP2D) and dosing frequency for Arm C will be selected by the SMG based on safety, tolerability, and PK / PD data collected during the dose escalation portion of the study.

[0164] TNB-486 dose escalation in combination with R-CHOP (Part 3, Arm D): In some embodiments, the methods involve evaluating the safety, tolerability, PK, and pharmacodynamic profile of TNB-486 in combination with R-CHOP administered every 3 weeks in approximately 9 to 18 subjects. The schematic study design for Part 3, Arm D, is shown in Figure 1B.

[0165] All subjects will receive six cycles of TNB-486 plus R-CHOP chemotherapy (cycles 1-6) at 21-day intervals, followed by three additional cycles of TNB-486 and rituximab (cycles 7-9) for a total of nine treatment cycles. R-CHOP will be administered according to standard of care at a fixed dose. In cycle 1, all subjects will receive R-CHOP on day 1, followed by two escalating step-up doses of TNB-486 on days 8 and 15. On day 1 of cycle 2, R-CHOP plus TNB-486 will be administered IV Q3W at the target dose. Dose escalation will begin at dose level (DL) 1 (i.e., TNB-486 270 μg on day 8 of cycle 1, followed by 1000 μg on day 15 of cycle 1; R-CHOP plus TNB-486 7200 μg on day 1 of cycle 2). Based on the DLT distribution, subsequent dose levels will be tapered or escalated to DL-1 or DL-2, respectively, as per Table 7.

[0166] [Table 7] Abbreviations: R-CHOP = rituximab, cyclophosphamide, doxorubicin, vincristine (Oncovin), prednisone.

[0167] Because common treatment-related toxicities of T cell engagers (i.e., CRS and NT) typically occur within 24-72 hours post-infusion, in some embodiments, the first subject in each cohort receives the target dose of TNB-486 + R-CHOP on Day 1 of Cycle 2 and then completes at least 1 week of observation, after which additional subjects may receive the target dose in that given cohort. In some embodiments, the DLT window is counted from Day 8 of Cycle 1 to Day 1 of Cycle 3 (a total of 3 TNB-486 doses, 35 days).

[0168] In some embodiments, subjects in Part 3, Arm D are enrolled according to an interval 3+3 (i3+3) design with a target toxicity rate of 30% and an equivalence interval (EI) of (25%-35%). In embodiments, up to three subjects may be enrolled simultaneously. In embodiments, the decision to de-escalate or escalate is made based on a minimum of three DLT-evaluable subjects per cohort. In embodiments, assuming three dose levels are investigated, approximately 9-18 subjects are enrolled, with 3-6 subjects assigned at each dose level. In embodiments, the total number of subjects depends on the number of dose escalations / de-escalations required.

[0169] In embodiments, once an active dose cohort of TNB-486 in combination with R-CHOP (i.e., a cohort in which at least one partial response or better is observed) is reached, and if the DLT rate of the highest dose cohort within EI (i.e., not requiring dose reduction) at the time of the SMG meeting has been achieved, active dose cohorts less than N may be expanded to up to 15 subjects each at the discretion of the SMG to better evaluate the optimal biologic dose / RP2D. In embodiments, if one or more dose levels are expanded, up to 15 subjects are assigned within each cohort.

[0170] In embodiments, if a DLT attributable to TNB-486 occurs according to investigator assessment, dosing will be withheld. TNB-486 treatment may be resumed at a lower dose only after toxicity has resolved to Grade 1 or less if, in the opinion of the treating investigator, the patient will derive clinical benefit. In embodiments, there will be no dose re-escalation of TNB-486 after recovery from toxicity, and intra-cohort participant dose escalation is not permitted. In embodiments, dose adjustments of the R-CHOP component follow conventional dose modification schedules.

[0171] Selection of study population: In some embodiments, patients undergo screening procedures within 28 days prior to receiving their first dose of study drug. Adult patients who meet the inclusion criteria and do not meet any of the exclusion criteria are eligible for enrollment in the study.

[0172] Inclusion Criteria: 1. Participants must be 18 years of age or older. 2. Subjects must have biopsy-proven B-NHL according to the World Health Organization (WHO) Criteria (Swerdlow 2017). 3. For Arm B only: Subjects must have biopsy-proven RR DLBCL or HGBL according to WHO Criteria (Swerdlow 2017). At least 15 subjects in Arm B must not have been previously treated with CD19-targeted therapy. 4. For Arm C only: At least 10 subjects in Arm C must not have been previously treated with a CD19-targeted therapy. 5. For Arm D only: Subjects have biopsy-proven, previously untreated CD20-positive DLBCL, including one of the following diagnoses per WHO Criteria (Swerdlow 2017): a.DLBCL, not otherwise specified (NOS) bT-cell / histiocytic-rich large B-cell lymphoma c. Epstein-Barr virus-positive DLBCL, NOS d.ALK-positive large B-cell lymphoma e. High-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements (double-hit or triple-hit lymphoma) f. High-grade B-cell lymphoma, NOS g. DLBCL transformed from indolent lymphoma h. Primary cutaneous DLBCL, leg type 1 i. Follicular lymphoma grade 3B. 6. Subject's B-NHL disease is CD19 positive. 7. Subjects have one or more measurable sites of disease as defined by the RECIL 2017 classification. (Younes 2017). 8. Subjects have received at least two lines of therapy to which they have been refractory or have subsequently relapsed. To be eligible for this study, subjects must not be candidates for treatment regimens known to provide clinical benefit in B-NHL. 9. For Arm D only: Subjects have not received prior treatment for the indication under study (except steroids for lymphoid symptom control) and are eligible to receive chemoimmunotherapy with R-CHOP in the opinion of the investigator. 10. For Arm D only: Subjects have an IPI score of 2-5. 11. For Arm D only: Subjects have a left ventricular ejection fraction (LVEF) within the institutional normal range as determined by cardiac echocardiogram (ECHO). 12. Subjects have an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less. 13. Subjects must have adequate bone marrow function defined as: 1000 / mm 3 Absolute neutrophil count (ANC) of 50,000 / mm or greater 3 Platelets >100,000 / mm for Part 3, Arm D 3 ≥ 8.0 g / dL), and hemoglobin ≥ 8.0 g / dL. Transfusion and / or growth factor support is permitted prior to evaluation, but for a subject to be eligible, neutrophils, platelets, and hemoglobin must be stable for at least 72 hours after transfusion and / or growth factor administration and prior to screening. If a subject receives a long half-life growth factor (e.g., pegylated granulocyte-colony stimulating factor (G-CSF)), the corresponding cell counts must be stable for at least 7 days for the subject to be eligible. 14. Subjects must have an estimated glomerular filtration rate (eGFR) of 50 mL / min or greater, as estimated by the MDRD equation. Note: Per FDA guidance, particularly for protein therapeutics >69 kDa, including antibodies such as TNB-486, "renal impairment is unlikely to alter PK sufficiently to justify a dosage adjustment" (FDA 2010). 15. Subjects must have total bilirubin less than 1.5 times the upper limit of normal (ULN; unless the subject has a known diagnosis of Gilbert's syndrome, in which case bilirubin must be less than 3 times the ULN). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) must be less than 3 times the ULN (unless the subject has a known diagnosis of Gilbert's syndrome, in which case AST / ALT must be less than 5 times the ULN).

[0173] Exclusion criteria: A subject is not eligible for treatment if they meet any of the following criteria: 1. Subject has been diagnosed with or treated for another malignant disease whose natural history or treatment may interfere with the safety or efficacy evaluation of the investigational regimen. Patients with a previous or concurrent malignant disease whose natural history or treatment may not interfere with the safety or efficacy evaluation of the investigational regimen are eligible for this trial. 2. Subject has a history of CNS disease due to B-NHL. If there is concern for CNS disease, an MRI and / or spinal tap will be performed at screening. 3. Subject has a history of leukemic manifestations of B-NHL, excluding MCL or MZL. 4. Subject has a history or presence of a clinically significant CNS pathology (e.g., epilepsy, seizures, stroke, paresis, aphasia, severe brain injury, dementia, neurodegenerative disorder (including Parkinson's disease, dementia, cerebellar disease, organic mental syndrome, psychosis, or other severe psychiatric illness)). 5. Subject has CNS lesions resulting from an active autoimmune disease or a history of autoimmune disease. 6. Subject experienced Grade 3 or greater CRS (based on ASTCT criteria) after prior TCE or CAR T-cell therapy. 7. Subject experienced Grade 2 or greater neurotoxicity / ICANS (based on ASTCT criteria) after prior TCE or CAR T-cell therapy. 8. The subject is a registered (of this drug) 1 / 2 received another investigational drug within 5 times the number of days or within 28 days, whichever is shorter. 9. Subject has undergone a peripheral autologous SCT within 12 weeks or an allogeneic SCT within 1 year of the first dose of study drug treatment, or has undergone an SCT and requires ongoing immunosuppressive therapy. 10. Subject requires chronic immunosuppressive therapy (including steroids >10 mg prednisone / day). For Part 3, Arm D, a steroid prephase is permitted. At the discretion of the medical monitor and sponsor, subjects may be eligible if immunosuppressive therapy is discontinued 14 days or 5 half-lives (whichever is shorter) before the first dose of study treatment. 11. Subject has any medical or psychiatric condition that, in the opinion of the investigator or medical monitor, may place the subject at an unacceptably high risk for toxicity, interfere with the successful or safe delivery of therapy, or interfere with the evaluation of the study drug or the subject's safety or interpretation of the study results. Examples include significant mucosal / internal bleeding, major psychiatric illness, substance abuse (including active alcoholism), active graft-versus-host disease, or a history of known allergy or hypersensitivity to any component of the study drug formulation. 12. Subject has received any therapy (including radiation, chemotherapy, biologics, and cell therapy) to treat cancer or major surgery within 14 days (or within 5 half-lives of anticancer drugs), whichever is shorter, prior to the first dose of study treatment. Administration of reduced-dose chemotherapy outside of this treatment holiday to reduce the risk of TLS or CRS / NT in subjects with bulky mass disease is permitted. 13. Subject has a known active infection requiring parenteral antibiotic treatment. Once parenteral antibiotics are completed and symptoms have resolved, the subject will be considered eligible for the study from an infection standpoint. 14. Subjects with human immunodeficiency virus (HIV) infection or chronic or active infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). HIV-infected patients on effective antiretroviral therapy who have an undetectable viral load within 6 months are eligible for this study. Subjects with chronic HBV may be enrolled if their HBV viral load is undetectable on suppressive therapy or if the subject is documented to be cured (hepatitis B surface antigen (HBsAg) negative). Subjects with HCV who are documented to be cured (HCV RNA undetectable 24 weeks after the end of treatment) may be enrolled. 15. Major cardiac abnormalities include, but are not limited to: uncontrolled angina or unstable life-threatening arrhythmias, history of myocardial infarction within 12 weeks prior to screening, New York Heart Association class 3 or greater congestive heart failure, severe heart failure, or persistent QTc prolongation (>480 msec, QTc Fridericia). 16. If female, subjects must not be pregnant, not lactating, and must be postmenopausal (for at least 12 consecutive months) or permanently surgically sterile, or for women of childbearing potential, must be using a protocol-specified highly effective method of female contraception (Section 0) beginning on Day 1 of Cycle 1 until at least 6 months after the last dose of study drug. 17. Subject has unresolved grade 2 or higher AEs from prior anticancer therapy, except for the following (National Cancer Institute [NCI] Common Terminology Criteria for Adverse Events [CTCAE] v5.0): - Alopecia. - Peripheral neuropathy (excluding grade 3 or higher peripheral neuropathy). - Anemia or thrombocytopenia (thrombocytopenia must be grade 4, grade 3 with symptoms or bleeding, or recur within 72 hours despite transfusion support to be excluded). - Subjects with irreversible toxicities (e.g., hearing loss) that are not reasonably expected to be exacerbated by the study drug may be included after consultation with the medical monitor. 18. For Arm D only: Current diagnosis of any of the following: a.Unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin lymphoma (gray zone lymphoma) b. Primary mediastinal (thymic) large B-cell lymphoma C. Burkitt lymphoma d.HHV8-positive DLBCL, NOS e. Primary effusion DLBCL 19. For Arm D: Contraindication to any of the individual components of R-CHOP, including prior administration of anthracycline. Contraception Recommendations and Pregnancy Testing: For women, subjects must be either postmenopausal, as defined as: · You are 55 years of age or older and have missed your period for 12 consecutive months or more without another medical cause. or Under 55 years of age, have missed menstruation for 12 consecutive months or more without another medical cause, and have a follicle-stimulating hormone level greater than 40 IU / L. or · Permanently surgically sterilized (bilateral oophorectomy, bilateral salpingectomy, or hysterectomy). or For women of childbearing potential, practicing a highly effective method of contraception from the list below, from Day 1 of Cycle 1 (or earlier) until at least 6 months after the last dose of study medication. - Initiation of combined (estrogen and progesterone-containing) hormonal contraception (oral, intravaginal, transdermal) associated with the inhibition of ovulation at least 1 month prior to Day 1 of Cycle 1. -Start progesterone-only hormonal contraception (oral, injectable, or implantable) that is associated with inhibition of ovulation at least 1 month prior to Day 1 of Cycle 1. - Bilateral tubal occlusion / ligation. - The partner has had a vasectomy and the vasectomized partner has a medical evaluation of surgical success and is the sole sexual partner of the study participant who is a woman of childbearing potential. - Condoms - Intrauterine contraceptive device. - Intrauterine hormone releasing system. - True abstinence: Abstinence from heterosexual intercourse when this is in line with the subject's preferred normal lifestyle (periodic abstinence [e.g., calendar, ovulation, symptom-temperature, post-ovulation methods] and extravaginal ejaculation are not permitted). For males, subjects of reproductive potential (i.e., male subjects who have not undergone bilateral vasectomies) must agree to use at least one highly effective method of male contraception (vasectomy or condoms) during sexual contact with females of childbearing potential, starting with the first dose of study medication and continuing until at least 6 months after the last dose of study therapy.

[0174] Therapeutic Administration: In some embodiments, TNB-486 is initially administered as an IV infusion Q2W, where one treatment cycle is 28 days. A minimum of 1 and a maximum of 9 subjects will be enrolled in Arm A at each dose level. A starting dose of 30 μg of TNB-486 will be administered as an IV infusion (Q2W) in the monotherapy dose escalation (Part 1, Arm A), escalating to a projected maximum of 30,000 μg in subsequent cohorts (Table 5). In Part 2, Arms B and C, all subjects will receive TNB-486 at the MTD and / or RP2D. Subjects may continue to receive TNB-486 until they meet the criteria for subject discontinuation.

[0175] In some embodiments, subjects are pre-medicated with dexamethasone (10 mg IV) or equivalent. If the subject does not experience an infusion-related reaction (IRR) or immune-mediated toxicity (IMT, e.g., CRS or NT), the dexamethasone pre-medication may be reduced to 5 mg IV. If the subject does not experience an IRR or IMT in a cycle in which they received 5 mg of dexamethasone IV as pre-medication, dexamethasone may be omitted from the pre-medication regimen thereafter. If the subject experiences an IRR or IMT at any time, or if the subject undergoes intra-subject dose escalation, the subject is pre-medicated with 10 mg of dexamethasone, and the next dose is TNB-486, tapering as described above.

[0176] In some embodiments, subjects are routinely premedicated with diphenhydramine (25-50 mg IV) or equivalent (e.g., cetirizine 10 mg orally [PO] × 1), acetaminophen 650-1000 mg PO, and ranitidine 150 mg PO / IV or equivalent 15-60 minutes prior to TNB-486 infusion to reduce the risk and severity of hypersensitivity reactions commonly observed with mAb therapy. Subjects may also be premedicated with tocilizumab (8 mg / kg IV) at the investigator's discretion and after approval by the medical monitor.

[0177] In some embodiments, the first TNB-486 infusion is given over 2 hours (±10 minutes). The duration of the infusion may be shortened after selection of an RP2D. Subjects will be hospitalized as inpatients for 48 hours after the infusion on Day 1 of Cycle 1. If the subject received a preparatory dose on C1D1 or experienced Grade 2 or higher CRS with the subject's first dose, the subject will also be hospitalized for 48 hours on C1D15. Subjects will be closely monitored for 2 hours after each infusion in subsequent cycles. For additional details regarding dose modifications, please refer to the Pharmacy Manual.

[0178] In some embodiments, the dose selected for evaluation in the monotherapy dose expansion (Part 2, Arms B and / or C) may be a dose at or below the MTD defined in the monotherapy dose escalation (Part 1, Arm A) or RP2D. The dosing frequency for the monotherapy dose expansion (Part 2, Arms B and / or C) will be selected by the sponsor, in consultation with the SMG, based on the safety, tolerability, and PK / PD data collected from the monotherapy dose escalation (Part 1, Arm A).

[0179] Dosage Form Preparation / Reconstitution: In some embodiments, TNB-486 drug product (active) is provided as a solution in a vial, formulated at 2 mg / mL with an extractable volume of 8 mL of drug product per vial, and administered via IV infusion. For the first four cohorts of monotherapy dose escalation (Part 1, Arm A; Table 5), TNB-486 is diluted in two steps. The first dilution step reduces the TNB-486 strength 100-fold to 20 μg / mL using the non-DEHP 50 mL IV bag provided with the kit. The second dilution step involves dose-dependently transferring a specified volume of pre-diluted TNB-486 into a non-DEHP 100 mL IV bag (100 mL non-DEHP-containing IV bag is provided by TeneoTwo). The final concentrations for the first four dose cohorts are 240 ng / mL, 720 ng / mL, 2.16 μg / mL, and 6.48 μg / mL, respectively. The diluent for each dilution step is normal saline to which IV stabilizer solution (IVSS) is added prior to adding the active TNB-486 drug product. IVSS is provided with each kit and consists of a 20 mL glass vial with an extractable volume of 15 mL. IVSS vials are formulated at 1X working strength and 10X strength in IV bags.

[0180] In some embodiments, test drug for dose cohorts 5 and above (Table 5) is prepared in a single dilution step, transferring dose-dependent volumes of TNB-486 directly from the drug product vial into the 100 mL non-DEHP IV bag provided with each kit. The diluent is saline to which IVSS is added prior to the addition of the active TNB-486 drug product, which is identical in formulation compared to the lower dose cohorts. The concentration of the active drug product ranges from 24.3 μg / mL to 303.7 μg / mL for dose cohorts 5 and above.

[0181] For all cohorts, the total storage time (including infusion time) of IV bags containing the final dilution of TNB-486 at controlled room temperature (20-25°C) should not exceed 6 hours (or 24 hours at 2-8°C) to minimize the risk of drug product degradation and microbial contamination. Storage times may be updated as additional sterility / stability data become available.

[0182] In some embodiments, the total volume administered for each dose is 250 mL. The infusion rate is controlled by a respective DEHP-free infusion set, which includes an infusion pump and an in-line filter.

[0183] In some embodiments, TNB-486 drug product vials are stored at 5±3° C. IVSS vials are stored at ambient temperature. The diluted active drug preparation was tested for infusion set compatibility at controlled room temperature (20-25° C.) for up to 6 hours and at 2-8° C. for up to 24 hours, with exposure to light, at the lowest dose (240 ng / mL) and the highest dose (303.7 μg / mL).

[0184] Eastern Cooperative Oncology Group (ECOG) performance status: Embodiments of the present disclosure include assessing the subject's ECOG performance status at various time points throughout the study. ECOG performance status will be recorded using the scoring system in Table 8.

[0185] [Table 8]

[0186] Pregnancy Test: Aspects of the present disclosure include obtaining serum or urine for pregnancy testing of women of childbearing potential at the time of central laboratory screening, where the serum or urine pregnancy test must be performed at a specific location within 72 hours of dosing and have a negative result. A urine pregnancy test (with confirmation by a serum pregnancy test if positive) must then be performed at a specific location and documented as negative before the start of dosing for each cycle.

[0187] Laboratory tests: Embodiments of the present disclosure include obtaining samples for the clinical tests outlined in Table 9 at specific locations at least at screening, and at subsequent visits, the EOT visit, and the 90-day follow-up visit as outlined in the Schedule of Events (all arms).

[0188] In some embodiments, a certified laboratory is utilized to process and provide the results of the clinical test. Laboratory reference ranges are obtained prior to the start of the study. The clinical test results for the clinical evaluation of a particular study are defined as the last measurement before the initial administration of TNB-486.

[0189] [Table 9] Abbreviations: Ab = antibody; aPTT = activated partial thromboplastin time; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; GGT = gamma-glutamyltransferase; HBsAg = hepatitis B surface antigen; HCV = hepatitis C virus; IFN = interferon; IL = interleukin; LDH = lactate dehydrogenase; MCH = mean corpuscular hemoglobin; PT = prothrombin time; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cells; TNF = tumor necrosis factor; WBC = white blood cells;

[0190] Biomarker assessment: Image Rating: In embodiments of the present disclosure, a baseline disease assessment using PET-CT, or CT in cases of non-FDG-active disease, must be performed within 28 days prior to the first dose of study drug for all subjects. In embodiments, imaging is repeated on the first day of every third cycle and as clinically indicated. Disease assessment may be performed by a central imaging provider. See Table 10 for details and examples of timing of disease response assessments.

[0191] Tumor tissue: In embodiments of the present disclosure, at screening, all subjects are required to provide 14-15 unstained tissue sections (e.g., from needle cores, resections, or fine-needle aspirates; if possible, at least 10 on charged slides and 4-5 on uncharged slides) or, if available, a formalin-fixed, paraffin-embedded block containing enough tumor to cut 14-15 sections from their most recent biopsy. The date of collection and a complete pathology report (including flow cytometry, cytogenetic / FISH, and molecular testing results, if performed) are to be submitted with the specimen. If the subject has previously received anti-CD19 therapy, a biopsy collected after disease recurrence or reoccurrence after CD19-targeted therapy is to be sent. In embodiments, tumor samples are analyzed at the molecular and cellular levels to determine how baseline biomarker levels and changes from baseline relate to clinical outcomes, safety, and tolerance.

[0192] In some embodiments, subjects in Part 2, Arms B and C will be asked to provide optional fresh tumor biopsies at screening, Cycle 3 Day 1, and at progression to evaluate exploratory biomarkers (e.g., next-generation sequencing to identify druggable mutations). In embodiments, the goal is to enroll 10 or more subjects who consent to these exploratory biomarker analyses in addition to the mandated biopsies / aspirations at the time of suspected CR and, if possible, at the time of suspected progression.

[0193] In some embodiments, subjects in Part 2, Arms B and C may be asked to provide optional fresh tumor biopsies at screening, Cycle 3 Day 1, and at progression to evaluate exploratory biomarkers (e.g., next-generation sequencing to identify druggable mutations). In embodiments, the goal is to enroll 10 or more subjects who consent to these exploratory biomarker analyses in addition to the mandated biopsies / aspirations at the time of suspected CR and, if possible, at the time of suspected progression.

[0194] [Table 10] Abbreviations: CR = complete response; EOT = end of treatment; PD = progressive disease; PET-CT = positron emission tomography-computed tomography. a Subjects in arms B and C will be asked to provide an optional fresh tumor biopsy at screening, C3D1, and when progression is suspected. b Subjects who discontinue treatment for reasons other than disease progression or withdrawal of consent will continue to undergo disease response assessments every 12 weeks (± 3 weeks) for the first 12 months, and every 26 weeks (± 4 weeks) thereafter until documentation of disease progression, initiation of subsequent anticancer therapy, or withdrawal. c The EOT visit will occur within 30 days after the last dose of TNB-486. If an alternative therapy is initiated during this period, the EOT visit will occur before the first dose of the alternative therapy. Adverse events and concomitant medications / therapies will be followed for 90 days after the last dose of study drug or until the subject starts a new therapy of choice, whichever comes first. Subjects will be followed for survival by phone every 12 weeks after the last visit. Once reliable half-life data for TNB-486 are available, T 1 / 2 If it is more than 18 days, T 1 / 2The 90-day post-last-treatment visit may occur further from the last dose to capture PK and ADA data at a T of 5 or more times greater than 90 days after the last dose. If this occurs, AEs will be collected until the time of this final visit or until a new course of therapy is initiated, whichever occurs first. d If bone marrow involvement due to the subject's disease is suspected at screening without a previous history, a bone marrow biopsy will be performed. If CR is suspected in a subject with a history of bone marrow involvement and / or if a biopsy is clinically indicated while the subject is on study, a bone marrow sample will be provided to a central laboratory for disease response assessment. e Disease assessment is performed centrally.

[0195] Blood samples: In embodiments of the present disclosure, blood samples will be collected from all subjects at the time points indicated in the event schedule (all arms) to assess PK, PD, and response biomarkers, as well as antidrug antibodies (ADA).

[0196] In some embodiments, blood samples are also collected at designated time points throughout the study to assess biomarkers (i.e., cytokines and exploratory biomarkers [e.g., circulating free DNA]) (Table 11). In addition, any remaining tissue or fluid samples taken from the subject during study or standard of care treatment may be used for assessment of exploratory biomarkers, with subject permission.

[0197] [Table 11] Abbreviations: ADA = anti-drug antibodies; EOI = end of infusion; EOT = end of treatment; min = minutes; N / A = not applicable; PK = pharmacokinetics; Unsched = unscheduled. f Samples collected at the same time point will be included in a single blood draw, if possible. Blood samples for PK, biomarker evaluation, and ADA testing will be shipped to a central laboratory. gPharmacogenomics is optional and should only be collected at screening. Separate consent required. h Tests for PK, ADA, and biomarkers (e.g., cytokines, circulating free DNA) will be batch analyzed centrally. Additional ADA testing will be performed during PK sampling timepoints as appropriate. i Unscheduled visits may occur at any time during the study. Indicated study activities will be performed at the discretion of the investigator. j Sufficient half-life data will be available for TNB-486, 1 / 2 If it is more than 18 days, T 1 / 2 To capture PK and ADA data at T > 5 times higher than T, the 90-day visit after the last treatment was 1 / 2 The time may be about five times the time required for the reaction.

[0198] ADA will be collected only on day 1 of each cycle except for cycle 1, where ADA samples will also be collected on day 15.

[0199] Exploratory research samples: In aspects of the present disclosure, serum samples for biomarker analysis are collected from all subjects.

[0200] Residual blood and tissue samples: In aspects of the present disclosure, residual blood and tissue samples previously collected for biomarker, PK, and tissue samples may be used for optional exploratory studies.

[0201] Pharmacogenomics samples: In embodiments of the present disclosure, an optional whole blood sample for DNA and RNA isolation is collected at screening or pre-dose on Day 1 of Cycle 1.

[0202] Activity, pharmacokinetics, pharmacodynamics, immunogenicity, pharmacogenetics, and safety assessments / variables: Active variables: In embodiments of the present disclosure, activity is measured by changes in target disease according to RECIL 2017 standards (see Table 12).

[0203] In some embodiments, activity endpoints (determined using RECIL 2017 response criteria) include objective response rate (ORR defined as CR+PR), clinical benefit rate (CBR defined as CR+PR+MR+SD at 24 weeks), overall survival (OS), progression-free survival (PFS), time to disease progression (TTP), time to response (TTR), and duration of objective response (DOR).

[0204] [Table 12] Abbreviations: CR = complete response; CT = computed tomography; FDG-PET = [18F]2-fluoro-2-deoxy-D-glucose; PR = partial response.

[0205] Pharmacokinetic variables: In an embodiment of the present disclosure, C max , C max Time to (T max ), the area under the concentration-time curve from time 0 to the time of the last measurable concentration (AUC t ), CL, terminal phase elimination rate constant, and t 1 / 2 The values ​​of PK parameters of TNB-486, including: PK parameters of TNB-486, will be determined after infusion in Cycle 1 using non-compartmental methods. Additional analyses will be performed if deemed useful and appropriate.

[0206] Drug concentration determination: In embodiments of the present disclosure, the time of collection of each blood sample will be recorded to the nearest minute on the source document and in the appropriate eCRF. In embodiments, the date and start / end times of the subject's TNB-486 infusion will also be recorded to the nearest minute on the day PK sampling occurs in the appropriate eCRF.

[0207] Pharmacokinetic time points may be slightly modified for monotherapy dose expansion (Part 2, Arms B and C) based on PK data from monotherapy dose escalation (Part 1, Arm A).

[0208] Blood sample collection for TNB-486 pharmacokinetic assay: In embodiments of the present disclosure, a single blood draw at a designated time point will allow for TNB-486 PK analysis for subjects in all arms of the study (see Table 13 and below). In embodiments, samples will not be drawn from the same arm in which TNB-486 is administered. In embodiments, for the Q2W dosing regimen, samples will be collected by venipuncture into appropriately labeled evacuated serum collection tubes at the following time points: (a) 12-week follow-up (Q2W) administration; (b) 12-week follow-up (Q2W) administration; (c) 12-week follow-up (Q2W) administration; (d) 12-week follow-up (Q2W) administration; (e) 12-week follow-up (Q2W) administration; (f) 12-week follow-up (Q2W) administration; (g) 12-week follow-up (Q2W) administration; (h) 12-week follow-up (Q2W) administration; (i) 12-week follow-up (Q2W) administration; (ii) 12-week follow-up (Q2W) administration; (iii) 12-week follow-up (Q2W) administration; (iv) 12-week follow-up (Q2W) administration; (v) 12-week follow-up (Q2W) administration; (vi ...

[0209] [Table 13]

[0210] Collection of blood samples for anti-drug antibody (ADA) assay: In an embodiment of the present disclosure, ADA samples are collected prior to dosing with TNB-486 on day 1 of each cycle and prior to dosing on day 15 of cycle 1.

[0211] Exploratory study variables: In some embodiments, exploratory studies are conducted to examine exposure-response relationships through biomarker relationships with PK, safety, and clinical activity. In embodiments, samples are collected to conduct exploratory research into known and novel biomarkers. The types of biomarkers analyzed include, but are not limited to, nucleic acids, proteins, lipids, or metabolites. Samples may be analyzed as part of a post-hoc evaluation of factors influencing a subject's response to a test drug or the development and progression of a subject's disease or related condition. Samples may also be used to develop new diagnostic tests, therapies, research methods, or technologies.

[0212] Safety variables: In embodiments of the present disclosure, adverse events, clinical laboratory profiles, physical examinations, and vital signs are assessed throughout the study. In embodiments, adverse events are graded according to NCI-CTCAE version 5.0.

[0213] Measurement validity: In embodiments of the present disclosure, standard PK, statistical, clinical, and laboratory procedures are utilized in this study. In embodiments, blood is also collected for PD markers, which may add useful information for selecting an appropriate dose of TNB-486 for future studies. Archived tissue may also be useful for selecting an appropriate dose of TNB-486 for future studies and for selecting an appropriate subject population for treatment.

[0214] Determining the maximum tolerated dose: In embodiments of the present disclosure, the MTD, if specified, is defined as the highest dose level at which fewer than 2 out of 6 subjects experience a DLT.

[0215] Determining the recommended Phase 2 dose: In embodiments of the present disclosure, if the MTD is reached, the RP2D is selected based on the totality of the data, rather than a dose higher than the MTD, by pooling and evaluating all available data regarding target engagement, clinical PK, PD, activity, and safety of TNB-486.

[0216] Toxicity control: In embodiments of the present disclosure, all AEs and laboratory abnormalities occurring during the study must be evaluated by the investigator for medical management purposes. The Clinical Toxicity Grade Table from NCI-CTCAE Version 5.0 (available on the CTEP homepage at http: / / ctep.info.nih.gov) will be used to grade AEs and laboratory abnormalities reported as AEs, each of which will be followed until satisfactory clinical resolution. TNB-486 has not been clinically tested in humans, and therefore the AE profile in humans is unknown.

[0217] In embodiments, AEs are classified as pre-existing or treatment-emergent (TEAE): A TEAE is defined as an AE not present before the initiation of TNB-486 treatment, or an AE present before the initiation of TNB-486 treatment that worsens in intensity and / or frequency after the initiation of TNB-486 treatment. A pre-existing AE is an AE that does not meet these criteria. Subjects with a pre-existing AE should be evaluated for any underlying disease or other cause and treated accordingly.

[0218] In embodiments, subjects with a TEAE should also be evaluated for concomitant illnesses or other causes and treated accordingly. If the TEAE is clearly attributable to the subject's underlying malignancy or other unrelated cause, the dosing of TNB-486 may be modified at the discretion of the principal investigator, in consultation with the medical monitor. Regardless of whether there is a "reasonable possibility" that the TEAE is TNB-486-related, if the TEAE is not clearly attributable to the subject's underlying malignancy or other unrelated cause, the dose of TNB-486 may be modified as follows: For subjects who complete Cycle 1 and have at least clinically or radiographically stable disease (SD, MR, PR, or CR) but experience a reversible TEAE, study drug dosing may be delayed for up to 28 days after the scheduled dosing date. Subjects who delay dosing for more than 28 days should be discontinued from the study. During any cycle, if a subject develops an ANC less than 500 / μL, platelets less than 10,000 / μL, or hemoglobin less than 6.5 g / dL, blood samples must be collected every 3 days and study treatment must be withheld. Treatment may be resumed if the ANC returns to ≥ 1,000 / μL, the platelet count returns to ≥ 50,000 / μL or baseline platelet count levels (in subjects with baseline platelets < 50,000 / μL), or the hemoglobin returns to ≥ 8.0 g / dL. In any subject experiencing CRS, TLS, or non-hematologic TEAEs that meet DLT-equivalent criteria, TNB-486 should be discontinued until toxicity is ≤ Grade 1 or has resolved to baseline. Study drug may then be resumed at a single reduced dose level with approval from the medical monitor.

[0219] In embodiments, up to two dose reductions are allowed to manage toxicity, after which the subject discontinues therapy if toxicity is deemed "unacceptable."

[0220] Although investigator discretion should be used in subject management for toxicity, guidelines for the management of CRS and NT are provided herein.

[0221] Dose-Limiting Toxicity (DLT) Definition for Dose Escalation: In embodiments of the present disclosure, the DLT observation period for dose escalation purposes is 28 days and covers the first complete treatment cycle of TNB-486 (two doses of TNB-486). Periodic conference calls are conducted between the SMG to review / confirm potential DLTs, evaluate AEs, and evaluate laboratory abnormalities. In embodiments, events occurring outside the DLT window may be evaluated during these calls when making dose escalation decisions. In embodiments, a DLT is defined as a TEAE that is not clearly attributable to the subject's underlying malignancy or other unrelated cause and meets the following criteria:

[0222] In aspects of the present disclosure, a DLT-evaluable subject is defined as a subject who: Subjects who receive at least one dose of TNB-486 and experience a DLT within the first 28 days or Subjects who receive at least two doses of TNB-486 on a Q2W schedule and are evaluated for toxicity during the 28-day evaluation period

[0223] In embodiments, NCI-CTCAE version 5.0 is used. DLT definitions are provided below.

[0224] Non-hematologic dose-limiting toxicities: Grade ≥ 3 non-hematologic AEs, excluding: - Isolated Grade 3 or 4 electrolyte abnormality (i.e., occurring without clinical consequences) that resolves to Grade 1 or less within 72 hours, with or without intervention. - Grade 3 hypoglycemia / hyperglycemia responding to optimal medical management within 72 hours. - Grade 3 nausea / vomiting / diarrhea that responds to optimal medical management within 72 hours. - Alopecia or vitiligo of any grade. - Grade 3 fatigue lasting less than 10 days. Any AE requiring a delay in the start of the next scheduled cycle of more than 28 days. Hepatotoxicity meeting Hy criteria.

[0225] Hematologic dose-limiting toxicities: Grade 3 CRS that does not resolve to Grade 1 or less within 72 hours or Grade 4 CRS. Grade 3 TLS that does not resolve to Grade 1 or less within 72 hours or Grade 4 TLS. Grade 4 neutropenia lasting more than 5 days or febrile neutropenia. Grade 3 thrombocytopenia with bleeding or Grade 4 thrombocytopenia. Grade 4 anemia. Grade 5 AE. Lymphopenia is not considered a DLT. Adverse events requiring a delay in the start of the next scheduled cycle of more than 28 days.

[0226] Cytokine Release Syndrome (CRS) dose-limiting toxicity: Cytokine release syndrome (CRS) is a major toxicity associated with T cell redirected therapy (CAR and T-BsAb / BiTE), regardless of the presence of neurotoxicity. CRS results from overactivation of the immune system and is primarily mediated by the secretion of proinflammatory cytokines, most importantly IL-6 and IL-1. Signs and symptoms are those of systemic inflammation and include: hyperthermia / rigors, hypotension, hypoxia, neurological changes, pain, nausea, and headache. CSR can present with varying severity, ranging from fever and flu-like symptoms to refractory hypotension and organ damage requiring high doses of vasopressors. Meta-analyses indicate that clinical findings, particularly fever, are usually the first indicator of CRS development (Hay 2017; Wang and Han 2018).

[0227] CRS historically occurs within 14 days of the first CAR / T-BsAb administration and usually does not occur in subsequent cycles. Low or selective activation by TNB-486 may delay CRS, if it occurs.

[0228] If CRS symptoms are suspected, grading should be performed to guide appropriate management. The consensus grading scheme published by Lee and colleagues is reproduced here and should be used to grade CRS (Table 14, Lee 2019). Figure 3 provides guidelines for treatments that may be used for subject management. However, if institutional guidelines for CRS management exist, investigators are encouraged to adhere to them.

[0229] [Table 14] Abbreviations: BiPAP = biphasic positive airway pressure; CPAP = continuous positive airway pressure; CRS = cytokine release syndrome. kFever is defined as a temperature of 38°C or higher not attributable to any other cause. In subjects with CRS who subsequently receive antipyretic or anticytokine therapy, such as tocilizumab or steroids, fever is no longer required to grade the severity of subsequent CRS. In this case, CRS grading is driven by hypotension and / or hypoxia. l CRS grade is determined by the more severe event: hypotension or hypoxia not attributable to any other cause. For example, a subject with a temperature of 39.5°C, hypotension requiring one vasopressor, and hypoxia requiring a low-flow nasal cannula is classified as having grade 3 CRS. m Low-flow nasal cannulae are defined as oxygen delivered at 6 L / min. Low-flow also includes blow-by oxygen delivery, which may be used in pediatric settings. High-flow nasal cannulae are defined as oxygen delivered at greater than 6 L / min. Source: Lee 2019

[0230] Neurological dose-limiting toxicities: The etiology of NT is unknown, but it is hypothesized to result from endothelial activation / microvascular injury, possibly downstream of IL-1 secretion by monocytes / macrophages (Gust 2017, Giavidris 2018, Norelli 2018). Onset usually occurs concomitantly with or after CRS (mostly CRS grade 3 or higher). Isolated NT has been described after administration of anti-CD19 T-BsAb (Velasquez 2017). Initial symptoms of NT include tremor, dysgraphia, expressive aphasia, attention deficits, and lethargy, which may be followed by delirium, headache, agitation, cerebral edema, ataxia, confusion, seizures, and coma.

[0231] If NT symptoms are suspected, grading should be performed to guide appropriate management. The consensus grading scheme published by Lee and colleagues is reproduced here and may be used to grade NT (Table 15, Lee 2019).

[0232] [Table 15] Abbreviations: EEG = electroencephalogram; ICANS = immune effector cell-associated neurotoxicity syndrome; ICE = immune effector cell-associated encephalopathy; ICP = intracranial pressure; NA = not applicable. NOTE: The ICANS grade is determined by the most severe event (ICE score, level of consciousness, seizures, motor findings, ICP elevation / cerebral edema) not attributable to any other cause. For example, a subject with an ICE score of 3 who has generalized seizures is classified as having grade 3 ICANS. a A subject with an ICE score of 0 can be classified as Grade 3 ICANS if they are arousable with total aphasia, whereas a subject with an ICE score of 0 can be classified as Grade 4 ICANS if they are not arousable. b Decreased level of consciousness should not be attributed to other causes (e.g., no sedatives). c Tremor and myoclonus associated with immune effector cell therapy may be graded according to CTCAE v5.0, but they do not affect ICANS grading. d Intracranial hemorrhage, with or without associated edema, is not considered a neurotoxicity feature and is excluded from ICANS grading, which may be graded according to CTCAE v5.0.

[0233] Source: Lee et al., 2019 Neurologic AEs in subjects receiving TNB-486 frequently require CAR-T cell therapy-associated toxicity (CARTOX-10) or immune effector cell encephalopathy (ICE) testing (Table 16) and neurologic examination. Early neurologic consultation, use of antiepileptic medications, and intensive care unit / airway support, if necessary, are encouraged. Table 17 provides guidelines for treatments that may be used in subject management, but investigators are encouraged to adhere to institutional guidelines for CRS management, if available.

[0234] [Table 16] Abbreviations: CARTOX-10 = chimeric antigen receptor toxicity; ICE = immune effector cell encephalopathy. Note: The CARTOX-10 (left column) has been updated to the ICE tool (right column). ICE adds a command-following assessment in place of one of the CARTOX-10 orientation questions. The scoring system remains the same. NOTE: Scoring: 10, no functional impairment; 7–9, Grade 1 ICANS; 3–6, Grade 2 ICANS; 0–2, Grade 3 ICANS; 0, Grade 4 ICANS due to subject being unable to arouse and perform an ICE assessment. Source: Lee 2019

[0235] [Table 17] Abbreviations: AE = adverse event; CT = computed tomography; EEG = electroencephalogram; ICU = intensive care unit; IV = intravenous; LP = lumbar puncture; MRI = magnetic resonance imaging.

[0236] Various dose-limiting toxicities: Any toxicity deemed related to the study drug will warrant drug withholding at the investigator's discretion. Other AEs may be considered DLTs, as determined by the TeneoTwo Medical Monitor in collaboration with the investigator.

[0237] All decisions regarding continued dosing for individual subjects will be medically managed by the Investigator in collaboration with the Medical Monitor, as appropriate. These decisions will be driven by the DLT criteria as described above.

[0238] Any subject who does not complete the full 28-day DLT observation period for any other than a DLT will be considered non-DLT-evaluable for dose escalation and / or MTD assessment and will be substituted at the same dose level. Additional subjects may be enrolled at a given dose level in the absence of a DLT to explore factors influencing AEs or to accumulate additional safety data.

[0239] Activity analysis: In embodiments of the present disclosure, activity data are listed for cohorts in monotherapy dose escalation (Part 1, Arm A). For monotherapy dose expansion (Part 2, Arms B and C), activity analyses will be performed based on the EE and safety populations, as appropriate. In embodiments, subject response and disease progression will be determined using RECIL 2017 criteria.

[0240] In embodiments of the present disclosure, objective response rate, DOR, PFS, and CBR are summarized and listed for both the monotherapy dose escalation (Part 1, Arm A) and the monotherapy dose expansion (Part 2, Arms B and C). Kaplan-Meier estimates for PFS and associated CIs for median PFS, OS, and TTP are provided. Additionally, in some embodiments, narrative summaries of ORR and its DOR and CBR, along with associated CIs, are provided. In embodiments, for the monotherapy dose expansion (Part 2, Arms B and C), activity analyses are performed based on the efficacy-evaluable population and repeated for the safety population unless the sample sizes of the two populations are the same. Additionally, in some embodiments, results from the monotherapy dose escalation (Part 1, Arm A) for the MTD or RP2D may be pooled with results from the monotherapy dose expansion (Part 2, Arms B and C) for analysis, as appropriate.

[0241] Objective response rate (ORR): In embodiments of the present disclosure, the objective response rate is defined as the proportion of subjects who have a confirmed partial or complete response to treatment, and may also be referred to as the overall response rate. In embodiments, the ORR for each dose cohort is estimated across all sites pooled. In embodiments, the two-sided 80% and 90% exact binomial CIs for the ORR, along with the best overall response (CR, PR, SD, PD), are also summarized using the Clopper-Pearson method.

[0242] Progression-free survival (PFS): In embodiments of the present disclosure, progression-free survival is defined as the time from the first dose of TNB-486 to progression or death, whichever occurs first. In embodiments, subjects are censored at the date of their last tumor assessment if neither event occurred. In embodiments, the Kaplan-Meier method is used to analyze PFS.

[0243] Duration of Objective Response (DOR): In embodiments of the present disclosure, the duration of a subject's objective response is defined as the time from the first objective response to disease progression or death, whichever occurs first. In embodiments, if the subject does not progress or die, the subject is censored at the date of last tumor assessment, similar to the censoring rules for PFS analysis.

[0244] In embodiments, DOR is analyzed in the same manner as PFS analysis.

[0245] Clinical benefit rate (CBR): In an embodiment of the present disclosure, the clinical benefit rate is defined as the proportion of subjects who have confirmed CR, PR, or MR, or SD for at least 24 weeks after responding to treatment. In an embodiment, the CBR of each arm is estimated for all sites pooled. In an embodiment, the two-sided 80% exact binomial CI of the CBR is also summarized using the Clopper-Pearson method.

[0246] Laboratory tests: In aspects of the present disclosure, baseline laboratory testing is performed at a central laboratory for the study. In aspects, all disease response assessment clinical trials from subsequent time points are performed at the central laboratory. In aspects, all samples for PK, ADA, and biomarker evaluation are also managed by the central laboratory. In aspects, all other clinical trials are performed at local laboratories associated with the clinical site.

[0247] In embodiments of the present disclosure, changes from baseline in clinical test results are summarized by dose cohort and time point using descriptive statistics. In embodiments, a summary of the shift from baseline to the last available visit is provided. In embodiments, the shift is calculated as the proportion of subjects at baseline who have values ​​below, within, or above the normal range for a particular clinical test compared to the proportion of subjects at the last visit who have values ​​below, within, or above the normal range.

[0248] In an embodiment of the present disclosure, laboratory abnormalities meeting NCI-CTCAE version 5.0 and treatment-emergent laboratory abnormalities are summarized by treatment arm and overall.

[0249] Eastern Cooperative Oncology Group (ECOG) Performance Score: In embodiments of the present disclosure, ECOG performance scores are listed in the subject data list and summarized by visit and change from baseline by visit.

[0250] Vital signs and physical examination: In aspects of the present disclosure, changes from baseline in vital signs and physical examination findings are summarized by dose cohort and time point using descriptive statistics.

[0251] Pharmacokinetics: Tables and summary statistics: In embodiments of the present disclosure, serum concentrations and PK parameter values ​​of TNB-486 are tabulated for each subject and each dose level, and summary statistics are calculated for each sampling time and each parameter.

[0252] Dose proportionality analysis: In an embodiment of the present disclosure, the pharmacokinetic parameters of TNB-486 from a particular dosing schedule evaluated on Day 1 of Cycle 1 are analyzed as follows: Analyses are performed for dose-normalized Cmax and dose-normalized AUC, provided that they can be appropriately determined from the data. The model used for statistical analysis includes TNB-486 dose level as a categorical variable. Covariates such as age, ethnicity, sex, and others that may potentially explain some of the variability in the population may be included in the initial model. However, if the regression coefficient is not significant at an alpha level of 0.10, the covariate may be removed from the model.

[0253] In embodiments, natural log transformations are used for Cmax and AUC unless the data clearly indicate that a different transformation or untransformed variables provides a more symmetric probability distribution and / or more uniform variance across dose levels. In embodiments, tests are performed on contrasts in dose-level effects chosen to be sensitive to an approximately linear function of dose or the logarithm of dose, assuming that at least three dose levels of TNB 486 are tested.

[0254] Further analysis will be conducted if useful and appropriate.

[0255] Missing values ​​and model violations: In embodiments of the present disclosure, all available data will be included in the dose-proportionality analysis. Any data exclusions will be documented and justified.

[0256] In this embodiment, the value of the PK variable (C max, AUC, etc.) are determined without imputing missing individual concentration values ​​by simply using available data. However, if a missing individual concentration would cause a PK parameter value to be too low or too high to be significant, the value of the PK parameter is considered provisionally missing. In this case, the missing individual concentration value is imputed so that the appropriate value of the PK parameter can be included in the analysis. In embodiments, the imputed value is obtained using an appropriate methodology that takes into account the individual characteristics of the subject.

[0257] In embodiments, outliers are identified and / or (C max If a significant non-normal probability distribution is observed (after logarithmic transformation of AUC and σ), non-parametric analyses can also be performed. Such model violations may be identified by graphical methods, measures of non-normality (e.g., skewness, kurtosis), or other appropriate methods. If different dose levels have unequal variances to an extent that may affect conclusions, an approximation method that allows for unequal variances will be used. Potential bias from missing data for subjects who discontinue early due to adverse events will be addressed.

[0258] Pharmacodynamics / biomarkers: In embodiments of the present disclosure, biomarker analysis is exploratory. In embodiments, descriptive statistics of baseline, post-baseline, and change from baseline biomarkers are summarized and listed by measurement time point / visit. In addition, exploratory analysis is performed to evaluate the association of each biomarker or combination of biomarkers with clinical outcomes, modulation of biomarkers related to mechanisms of action, and biomarkers or combinations of biomarkers that may predict treatment response.

[0259] V. Pharmaceutical Compositions for Use Embodiments of the present disclosure include a pharmaceutical composition for treating B-cell non-Hodgkin's lymphoma in a patient, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486, wherein the TNB-486 is administered according to a 28-day treatment cycle, and the therapeutically effective amount of TNB-486 in the pharmaceutical composition is from about 30 μg to about 30,000 μg.

[0260] In some embodiments of the pharmaceutical composition for use, the treatment cycle is repeated two or more times. In some embodiments, TNB-486 is administered to the patient as monotherapy. In some embodiments, TNB-486 is administered by intravenous infusion (IV). In some embodiments, the patient has received at least two prior systemic therapies. In some embodiments, the patient is CD19-positive. In some embodiments, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less. In some embodiments, the patient has adequate bone marrow function. In some embodiments, the patient has an estimated glomerular filtration rate (eGFR) of 50 mL / min or greater. In some embodiments, the patient has a total bilirubin level of 1.5 times the upper limit of normal, an aspartate aminotransferase (AST) level of 3 times the upper limit of normal, and an alanine aminotransferase (ALT) level of 3 times the upper limit of normal.

[0261] Embodiments of the present disclosure include a pharmaceutical composition for improving objective response rate (ORR) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0262] Embodiments of the present disclosure include a pharmaceutical composition for improving overall survival (OS) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0263] Embodiments of the present disclosure include a pharmaceutical composition for improving progression-free survival (PFS) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0264] Embodiments of the present disclosure include a pharmaceutical composition for improving time to disease progression (TTP) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0265] Embodiments of the present disclosure include a pharmaceutical composition for improving time to response (TTR) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0266] Embodiments of the present disclosure include a pharmaceutical composition for improving duration of objective response (DOR) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0267] Embodiments of the present disclosure include a pharmaceutical composition for improving clinical benefit rate (CBR) in patients diagnosed with B-cell non-Hodgkin's lymphoma, the pharmaceutical composition comprising a therapeutically effective amount of TNB-486 administered according to a 28-day cycle, the therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

[0268] In some embodiments of the pharmaceutical composition for use, the improvement is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

[0269] In some embodiments of the pharmaceutical composition for use, the treatment cycle is modified to add more time between doses, hi some embodiments, the treatment cycle is modified by consistently eliminating one or more treatment cycles from the dosing regimen.

[0270] In some embodiments of the pharmaceutical composition, the treatment cycle is amended to include a priming dose. In some embodiments, the priming dose is from about 150 μg to about 1500 μg. In some embodiments, the priming dose is from about 270 μg to about 1000 μg. In some embodiments, the priming dose is administered at the first time point in the first treatment cycle, and a full dose is administered at all subsequent time points. In some embodiments, the priming dose is administered on day 1 of the first treatment cycle, a full dose is administered on day 15 of the first treatment cycle, and a full dose is administered on days 1 and 15 of all subsequent treatment cycles.

[0271] In some embodiments of the pharmaceutical composition, a treatment cycle is modified to include at least two priming doses. In some embodiments, the first priming dose is about 150 μg to about 540 μg. In some embodiments, the second priming dose is about 800 μg to about 1200 μg. In some embodiments, the first priming dose is about 270 μg and the second priming dose is about 1000 μg. In some embodiments, the first priming dose is administered on day 1 of the first treatment cycle, the second priming dose is administered on day 8 of the first treatment cycle, a complete dose is administered on day 15 of the first treatment cycle, and a complete dose is administered on days 1 and 15 of all subsequent treatment cycles.

[0272] In some embodiments of the pharmaceutical composition, the full dose is no more than 100% greater than the full dose corresponding to the next lowest dose cohort. In some embodiments, the full dose is no more than 50% greater than the full dose corresponding to the next lowest dose cohort. In some embodiments, the full dose is no more than 33% greater than the full dose corresponding to the next lowest dose cohort.

[0273] In some embodiments of the pharmaceutical composition for use, the patient is pre-medicated with an agent that reduces the risk or severity of a hypersensitivity reaction prior to administration of TNB-486. In some embodiments, the agent that reduces the risk or severity of a hypersensitivity reaction is selected from the group consisting of dexamethasone, diphenhydramine, acetaminophen, ranitidine, tocilizumab, any equivalent thereof, or any combination thereof. In some embodiments, the agent that reduces the risk or severity of a hypersensitivity reaction is administered 15 to 60 minutes prior to administration of TNB-486. In some embodiments of the pharmaceutical composition for use, the therapeutically effective amount of TNB-486 is about 30 μg, 90 μg, 270 μg, 800 μg, 2400 μg, 7200 μg, 15000 μg, or 30000 μg.

[0274] Example 1 Results of a Phase 1 Study of TNB-486 in Patients with Relapsed / Refractory B-NHL background: TNB-486 is a novel CD19×CD3 bispecific T-cell engager (TCE) incorporating a unique anti-CD3 moiety designed to reduce cytokine release syndrome by binding to T cells with low affinity. The silenced IgG4 backbone provides a long half-life suitable for intermittent dosing. In DLBCL, combining TNB-486, which targets CD19, with R-CHOP, which incorporates Rituxan-mediated CD20 targeting, may result in synergistic tumor killing, reduce the risk of antigen escape, and offer a promising approach for improved long-term remission. Here, we present interim results from an ongoing first-in-human (FIH) phase 1 trial of TNB-486 in R / R 8-NHL.

[0275] method: The primary objectives of this study were to evaluate the safety, tolerability, and pharmacokinetics of TNB-486 when administered as monotherapy and to determine the optimal biologically active dose. Patients with relapsed / relapsed 8-NHL after at least two prior lines of therapy were eligible. Prior anti-CD19 therapy was permitted. Patients received escalating doses of TNB-486 administered via IV infusion over 1–2 hours Q2W until PD / unacceptable toxicity. Fixed doses were given initially at a low dose, followed by a preparatory dose at a higher target dose (>2.4 mg). Responses were assessed by RECIL 2017, and adverse events were graded using the CTCAE, with the exception of cytokine release syndrome (CRS) and neurotoxicity (NT), which were graded according to ASTCT criteria.

[0276] result: As part of the current study for the first clinical cutoff, 27 subjects received TNB-486 at doses ranging from 0.030 to 10 mg. Twenty-two patients were evaluable for efficacy and 27 were evaluable for safety. Patient characteristics are summarized in Table 18. Overall, patients were heavily pretreated, with a median of four prior lines of therapy (range 2 to 21 prior lines), and 19% of enrolled subjects had prior CAR-T failure.

[0277] Upon expanding enrollment and evaluating 30 patients for the second clinical cutoff, 25 were evaluable for efficacy and 30 were evaluable for safety. Patient characteristics are summarized in Table 19. For the second clinical cutoff, 23% of enrolled subjects had prior CAR-T failure.

[0278] [Table 18]

[0279] [Table 19]

[0280] For the first clinical cutoff, only one case of Grade 3 CRS was observed, and no CRS (of any grade) occurred after Cycle 1. The median time to CRS onset was 1 day, and the median time to resolution was 1.5 days (range, <1 to 9). Eight subjects received tocilizumab, and six of these had Grade 2 CRS. Six subjects (22%) experienced NT (7% Grade 3, none Grade 4, occurring in two subjects with MZL and Richter's transformation, respectively), all of which were transient and resolved without sequelae. No Grade 2 or higher NT was reported in DLBCL or FL, and no new cases of NT occurred beyond Cycle 1. Four subjects received steroids for NT. Notably, one subject who experienced Grade 3 NT continued therapy at a reduced dose and did not relapse.

[0281] CRS results were very similar at the second clinical cutoff, as shown in Table 20 and Figure 5A.

[0282] [Table 20]

[0283] Immune effector cell-associated neurotoxicity syndrome (ICANS) was assessed at a second clinical cutoff, and the results are shown in Table 21 and Figure 5B. Events classified as neurotoxicity were reported by 10 of 30 subjects (33%). All of these events resolved. Grade 3 events occurred in four patients with risk factors (e.g., age >80 years, aggressive histology, and / or previous G4 event after CART). Three of these four patients were subsequently rechallenged without recurrence.

[0284] [Table 21]

[0285] Preliminary PK data showed mean T ranged from 7.55 to 11.7 days at the active dose. 1 / 2, supporting Q2W (or less frequent) dosing of TNB-486.

[0286] First clinical cutoff: At the date of the first clinical cutoff, the overall response rate (ORR) at 800 μg was 72% (13 of 18 evaluable subjects), and the CR rate was 61% (11 of 18). Among response-evaluable CAR-T-exposed subjects, 2 / 3 achieved a CR. Among response-evaluable FL subjects treated with 800 μg, the ORR was 88% (7 of 8 subjects). All responding subjects achieved a CR. Five DLBCL subjects treated with 800 μg had an ORR of 40%. One subject achieved an MRD-negative CR. Notably, this subject had been previously treated with five prior therapies, including CAR-T, and had not achieved a CR prior to this study. Among four subjects with MZL, the ORR was 75% (all CR). With a median follow-up of 3.8 months (range: 1.5-8.7 months), no subjects in CR relapsed and remained in remission for up to 10 months after initiation of TNB-486 therapy (Figure 6). Responses were seen across all NHL subtypes and prognostic factors (e.g., disease burden, prior therapy, and refractoriness to prior therapy).

[0287] Second Clinical Cutoff: At the date of the second clinical cutoff, the overall response rate (ORR) was greater than 75% (19 of 25 evaluable subjects), and the CR rate was 63% (16 of 25). Among response-evaluable CAR-T-exposed subjects, 2 / 4 achieved a CR. Among response-evaluable FL subjects, the ORR was 88% (7 of 8 subjects), and all responding subjects achieved a CR. See Figure 7. With a median follow-up of 3.8 months (range: 1.5-8.7 months), subjects in CR did not relapse and remained in remission for up to 10 months after initiation of TNB-486 therapy (Figure 8). Responses were seen across all NHL subtypes and prognostic factors (e.g., disease burden, prior therapy, and refractoriness to prior therapy). Preliminary data from a recent FIH trial of TNB-486 demonstrate an acceptable safety profile with mostly low-grade CRS / NT at doses up to 7.2 mg (dose escalation is ongoing), and promising activity in heavily pretreated B-NHL.

[0288] Third Clinical Cutoff: At the third clinical cutoff date, 17 patients received TNB-486 at a target dose of 0.03–10 mg (median age 68 years [range 33–86], 53% male, 65% stage III / IV disease, 25% CD20 disease, median prior lines of therapy (LOT) 3 [range 2–9]). Prior therapy included αCD20 Ab (100%), alkylating agents (76%), IMiD (47%), CD20 TCE (12%), CD19 CAR T (12%), and ASCT (6%). 53% progressed or initiated a second LOT within 24 months of initiation of the first LOT (POD24). Median study duration was 7 months (range 1–22). Eleven patients were evaluable for efficacy at a target dose of 2.4 mg or higher. As shown in Figure 10, the objective response rate (ORR) and complete response rate (CR) were 91%. The ORR / CR rate for patients with CD20 disease, prior CD20 TCE, and POD 24 was 100%. Of patients with a CR, one patient with six prior LOTs progressed at C6 and maintained CD19 expression. The 6-month PFS rate was 91%. No G3+ CRS occurred (59% G1, 12% G2). Neurological events consistent with ICANS were reported in 24% of patients, with one G3 event (confusion). All CRS / NT were transient and resolved within a median of 1.5 days (range 1-5). G3+ treatment-related AEs in more than 10% of patients included lymphopenia (35%) and neutropenia (12%).

[0289] Example 2 Further results from Phase I trials This example provides additional data obtained during Part 1, Arm A of the Phase 1 study described above in Example 1. A summary of the Arm A cohort is provided in Table 22.

[0290] [Table 22]

[0291] For each cohort, overall response, objective response rate, and clinical benefit rate were assessed as described above. A summary of responses for each cohort is shown in Table 23. Abbreviations are as defined above.

[0292] [Table 23] [1] Best overall response is defined for each subject as the best response observed after treatment and before the initiation of any new anticancer therapy. [2] ORR is defined as the proportion of subjects with a confirmed partial or complete response to treatment as determined by RECIL 2017. [3] CBR is defined as the proportion of subjects who experience a confirmed CR, PR, or MR, or SD for at least 24 weeks after responding to treatment, as determined by RECIL 2017.

[0293] A summary of responses for all fixed and single priming dose cohorts, all double step-up priming dose cohorts, and all subjects combined is provided in Table 24. Abbreviations are as defined above and are the same as in Table 23.

[0294] [Table 24] [1] Confidence intervals are based on Clopper-Pearson confidence limits.

[0295] Outcomes in each cohort were also determined based on B-cell non-Hodgkin's lymphoma (B-NHL) subtype. Results for all fixed-dose and single-primary-dose cohorts by B-NHL subtype are summarized in Table 25. Abbreviations used in the following tables are as follows: DLBCL / HGBL = diffuse large B-cell lymphoma / high-grade B-cell lymphoma; FL = follicular lymphoma; MZL = marginal zone lymphoma; MCL = mantle cell lymphoma.

[0296] [Table 25]

[0297] Results by subtype for double step-up priming dose cohorts 5d and 6d are shown in Tables 26 and 27.

[0298] [Table 26]

[0299] [Table 27]

[0300] Target doses of 2400 μg and above were found to have significant ORR and CBR. A summary of results by B-NHL subtype for all doses above 2400 μg is provided in Table 28.

[0301] [Table 28]

[0302] Cytokine release after drug administration was measured for the following cytokines: interleukins (IL-2, IL-6, IL-8, and IL-10); interferon gamma (INFγ); monocyte chemoattractant protein-1 (MCP-1); macrophage inflammatory protein-1 alpha (MIP-1α); and tumor necrosis factor alpha (TNFα). Cytokine release was not observed for IL-2, IL-6, MIP-1α, INFγ, and TNFα in any cohort. Some release of IL-8 and IL-10 was observed, which generally resolved after 9 to 24 hours. Release of MCP-1 was seen in all cohorts, but also resolved after 9 to 24 hours for most doses.

Claims

1. 1. A method for treating B-cell non-Hodgkin's lymphoma (B-NHL) in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to 28-day treatment cycles, said therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg, and optionally said patient receiving six treatment cycles.

2. 10. The method of claim 1, wherein the treatment cycle is repeated two or more times.

3. 3. The method of claim 1 or 2, wherein TNB-486 is administered to said patient as monotherapy.

4. The method of any one of claims 1 to 3, wherein TNB-486 is administered by intravenous infusion (IV).

5. The method of any one of claims 1 to 4, wherein the patient has received at least two prior systemic therapies.

6. The method of any one of claims 1 to 5, wherein the patient's B-NHL disease is CD19 positive.

7. 7. The method of any one of claims 1 to 6, wherein the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less.

8. The method of any one of claims 1 to 7, wherein the patient has adequate bone marrow function.

9. The method of any one of claims 1 to 8, wherein the patient has an estimated glomerular filtration rate (eGFR) of 50 mL / min or greater.

10. The patient is Total bilirubin is less than 1.5 times the upper limit of normal, aspartate aminotransferase (AST) less than three times the upper limit of normal; The method according to any one of claims 1 to 9, wherein alanine aminotransferase (ALT) is not more than three times the upper limit of normal.

11. 1. A method of improving objective response rate (ORR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, said therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

12. 1. A method for improving overall survival (OS) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein said therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

13. 1. A method for improving progression-free survival (PFS) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, said therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

14. 1. A method for improving time to disease progression (TTP) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein said therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

15. 1. A method for improving time to response (TTR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day treatment cycle, wherein said therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

16. 1. A method for improving duration of objective response (DOR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day cycle, wherein said therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

17. 1. A method for improving clinical benefit rate (CBR) in a patient diagnosed with B-cell non-Hodgkin's lymphoma, said method comprising administering to said patient a therapeutically effective amount of TNB-486 according to a 28-day cycle, said therapeutically effective amount of TNB-486 being from about 30 μg to about 30,000 μg.

18. 18. The method of any one of claims 11 to 17, wherein the improvement is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

19. 19. The method of any one of claims 1 to 18, wherein the treatment cycle is modified to add more time between doses.

20. 19. The method of any one of claims 1 to 18, wherein the treatment cycle is modified by consistently eliminating one or more treatment cycles from the dosing regimen.

21. 19. The method of any one of claims 1 to 18, wherein the treatment cycle is modified to include a priming dose.

22. 22. The method of claim 21, wherein the priming dose is from about 150 μg to about 1500 μg.

23. 22. The method of claim 21, wherein the priming dose is from about 270 μg to about 1000 μg.

24. 23. The method of claim 21 or 22, wherein the priming dose is administered at a first time point in a first treatment cycle and a full dose is administered at all subsequent time points.

25. 24. The method of claim 23, wherein the priming dose is administered on day 1 of the first treatment cycle, the full dose is administered on day 15 of the first treatment cycle, and the full dose is administered on days 1 and 15 of all subsequent treatment cycles.

26. 22. The method of claim 21, wherein the treatment cycle is modified to include at least two priming doses.

27. 27. The method of claim 26, wherein the first priming dose is from about 150 μg to about 540 μg.

28. 28. The method of claim 26 or 27, wherein the second priming dose is from about 800 μg to about 1200 μg.

29. 29. The method of any one of claims 26-28, wherein the first priming dose is about 270 μg and the second priming dose is about 1000 μg.

30. 30. The method of any one of claims 26-29, wherein the first priming dose is administered on day 1 of a first treatment cycle, the second priming dose is administered on day 8 of the first treatment cycle, a full dose is administered on day 15 of the first treatment cycle, and the full doses are administered on days 1 and 15 of all subsequent treatment cycles.

31. 31. The method of claim 24, 25, or 30, wherein the full dose is no more than 100% greater than the full dose corresponding to the next lowest dose cohort.

32. 31. The method of claim 24, 25, or 30, wherein the full dose is no more than 50% greater than the full dose corresponding to the next lowest dose cohort.

33. 31. The method of claim 24, 25, or 30, wherein the full dose is no more than 33% greater than the full dose corresponding to the next lowest dose cohort.

34. 34. The method of any one of claims 1 to 33, further comprising pre-medicating the patient with an agent that reduces the risk or severity of a hypersensitivity reaction prior to administration of TNB-486.

35. 35. The method of claim 34, wherein the agent that reduces the risk or severity of a hypersensitivity reaction is selected from the group consisting of dexamethasone, diphenhydramine, acetaminophen, ranitidine, tocilizumab, any equivalents thereof, or any combination thereof.

36. 46. ​​The method of claim 34 or 45, wherein the agent that reduces the risk or severity of a hypersensitivity reaction is administered 15 to 60 minutes prior to administration of TNB-486.

37. 37. The method of any one of claims 1 to 36, wherein the therapeutically effective amount of TNB-486 is about 30 μg, 90 μg, 270 μg, 800 μg, 2400 μg, 7200 μg, 15000 μg, or 30000 μg.

38. 38. The method of any one of claims 1 to 37, wherein the TNB-486 is administered in combination with another chemotherapy.

39. 39. The method of claim 38, wherein the additional chemotherapy is a combination of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunomycin), vincristine sulfate (Oncovin), and prednisone (R-CHOP).

40. 40. The method of claim 39, wherein the R-CHOP is administered on day 1 of the first treatment cycle, and then on day 1 of each subsequent treatment cycle.

41. 40. The method of claim 39, wherein each treatment cycle is 21 days long, R-CHOP is administered on day 1 of a first treatment cycle, a first priming dose is administered on day 8 of said first treatment cycle, a second priming dose is administered on day 15 of said first treatment cycle, and R-CHOP and a therapeutically effective amount of TNB-486 are administered on day 1 of a second treatment cycle.

42. 42. The method of claim 41, wherein R-CHOP and a therapeutically effective amount of TNB-486 are administered on day 1 of each subsequent treatment cycle.

43. 43. The method of any one of claims 21 to 42, wherein the patient has reduced cytokine release compared to a treatment cycle without a priming dose.

44. 44. The method of any one of claims 21 to 43, wherein the patient does not experience cytokine release syndrome or only experiences grade 1 cytokine release syndrome.

45. 45. The method of claim 43 or 44, wherein the cytokine release syndrome comprises release of IL-6 and / or TNF-α.

46. 46. ​​The method of any one of claims 1 to 45, wherein the objective response rate (ORR) is 80% or more, or the 6-month PFS rate is 90% or more, or the complete response (CR) rate is 90% or more.

47. 47. The method of any one of claims 1 to 46, wherein TNB-486 is a bispecific molecule that binds to CD3 and human CD19, and comprises: (i) a first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 18; (ii) a second polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 11, wherein the first polypeptide subunit and the second polypeptide subunit together form a first binding moiety that binds to human CD3; and (iii) a third polypeptide subunit that binds to human CD19, comprising the amino acid sequence of SEQ ID NO: 20.

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