Methods of treating non-hodgkin lymphoma

Abstract

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

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 381,886, filed on November 1, 2022, and U.S. Provisional Application No. 63 / 504,179, filed on May 24, 2023, under 35 U.S.C. § 119(e). Both applications are hereby incorporated by reference in their entirety for all purposes.

[0003] Reference to Sequence Listing

[0004] This application incorporates by reference the Sequence Listing submitted together with this application as an HTML file named CD19TCE - 201 - WO - PCT, created on October 31, 2023, and having a size of 28.0 kilobytes. Technical Field

[0005] The present disclosure provides methods of treating non - Hodgkin lymphoma by administering a multispecific (e.g., bispecific) antibody to a patient in need thereof. The present disclosure also provides methods of preparing such antibodies, and compositions comprising such antibodies, including pharmaceutical compositions. Background Art

[0006] The incidence rate of new cases of non - Hodgkin lymphoma (NHL) is 19.6 per 100,000 men and women per year. The mortality rate is 5.4 per 100,000 men and women per year. These rates are age - adjusted and are based on cases and deaths from 2014 to 2018. The estimated number of new cases in 2021 was 81,560; representing 4.3% of all new cancer cases. The estimated number of new deaths in 2021 was 20,720; representing 3.4% of all cancer deaths. At presentation, 24% of cases are stage I defined as "confined to a single region", 14% of cases are stage II defined as "involving multiple regions", 16% of cases are stage III defined as "spread to both sides of the diaphragm", 33% of cases are stage IV defined as "diffuse or disseminated involvement", and 12% are unstaged. The 5 - year relative survival rate is 84.3% for stage I, 77.1% for stage II, 71.1% for stage III, and 63.7% for stage IV. SEER database; accessed on April 15, 2021; https: / / seer.cancer.gov / .

[0007] Non-Hodgkin lymphoma includes diverse tumors of the lymphoid compartment; overall, approximately 74,000 new cases are reported annually in total (National Cancer Institute [NCI] Surveillance, Epidemiology, and End Results [SEER] 2020). In the United States (US), approximately 80% of these are derived from the B-cell lineage. Among B-cell non-Hodgkin lymphoma (B-NHL), subtypes can be loosely grouped into 'indolent' lymphomas, including chronic lymphocytic leukemia / small lymphocytic lymphoma (not studied in this investigation), 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 lymphomas; additional types and subtypes exist (Swerdlow 2017).

[0008] Diagnosis typically includes techniques such as physical examination, blood and urine tests, imaging, and lymph or bone marrow tests. Conventional therapies include chemotherapy administered orally or by injection, radiotherapy, and bone marrow transplantation.

[0009] For DLBCL, more than half of patients survive 5 years or longer using multi-agent chemotherapy regimens that include rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (e.g., R-CHOP; Friedberg 2011). Second-line therapy typically consists of high-dose chemotherapy and subsequent autologous SCT. For patients not suitable for transplantation, a wide range of therapies that usually incorporate anti-CD20 monoclonal antibodies are used. Only 30% to 40% of these respond (Crump 2017). Patients who do not respond to salvage therapy have a poor prognosis. Third-line therapies include anti-CD19 chimeric antigen receptor (CAR) T cells (e.g., and ) and anti-CD79b ADC (Yescarta US Prescribing Information [PI] 2017; Kymriah USPI 2017; Polivy USPI 2019). The eligibility for second-line and third-line therapies varies greatly for patients. For example, elderly or frail patients are generally not candidates for transplantation, and up to 23% of patients with relapsed or refractory (RR) DLBCL have disease progression too rapid to be eligible for CAR T-cell therapy (Paillassa 2019). In addition, none of the above third-line therapies are currently considered 'available therapies'. Except for second-line, overall survival (OS) is in the range of 8 to 10 months (Van Den Neste 2016). Given these characteristics, DLBCL (especially RR or high-risk disease) represents one of the most important areas of unmet medical need in lymphoma.

[0010] Aspects of the present disclosure include methods of treating this patient population. Summary of the Invention

[0011] Aspects of the present disclosure include methods for treating B-cell non-Hodgkin lymphoma in a patient in need thereof, the methods 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, optionally wherein the patient receives six treatment cycles.

[0012] In some aspects, the treatment cycle is repeated two or more times. In some aspects, TNB-486 is administered to the patient as a single therapy. In some aspects, TNB-486 is administered by intravenous infusion (IV). In some aspects, the patient has received at least two prior lines of systemic therapy. In some aspects, the patient is CD19 positive. In some aspects, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of less than or equal to 2. In some aspects, the patient has adequate bone marrow function. In some aspects, the patient has an estimated glomerular filtration rate (eGFR) of greater than or equal to 50 mL / min. In some aspects, the subject has a total bilirubin less than or equal to 1.5 times the upper limit of normal, an aspartate aminotransferase (AST) less than or equal to 3 times the upper limit of normal, and an alanine aminotransferase (ALT) less than or equal to 3 times the upper limit of normal.

[0013] Aspects of the present disclosure include methods for improving the objective response rate (ORR) in a patient diagnosed with B-cell non-Hodgkin lymphoma, the methods 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] Aspects of the present disclosure include methods for improving the overall survival (OS) rate of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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] Aspects of the present disclosure include methods for improving the progression-free survival (PFS) rate of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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] Aspects of the present disclosure include methods for improving the time to progression (TTP) of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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.

[0017] Aspects of the present disclosure include methods for improving the time to response (TTR) of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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.

[0018] Aspects of the present disclosure include methods for improving the duration of objective response (DOR) of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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.

[0019] Aspects of the present disclosure include methods for improving the clinical benefit rate (CBR) of patients diagnosed with B-cell non-Hodgkin lymphoma, the methods including 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.

[0020] In some aspects, 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%.

[0021] In some aspects, the treatment cycle is modified to increase the amount of time between doses. In some aspects, the treatment cycle is modified by consistently eliminating one or more treatment cycles from the dosing regimen.

[0022] In some aspects, the treatment cycle is modified to include a priming dose. In some aspects, the priming dose is from about 150 μg to about 1500 μg. In some aspects, the priming dose is from about 270 μg to about 1000 μg. In some aspects, the priming dose is administered at a first time point in the first treatment cycle and the full dose is administered at all subsequent time points. In some aspects, 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 day 1 and day 15 of all subsequent treatment cycles.

[0023] In some aspects, the treatment cycle is modified to include at least two priming doses. In some aspects, the first priming dose is from about 150 μg to about 540 μg. In some aspects, the second priming dose is from about 800 μg to about 1200 μg. In some aspects, the first priming dose is about 270 μg and the second priming dose is about 1000 μg. In some aspects, 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, the full dose is administered on day 15 of the first treatment cycle, and the full dose is administered on day 1 and day 15 of all subsequent treatment cycles.

[0024] In some aspects, the full dose is equal to 100% of the full dose corresponding to the next lowest dose group or is more than 100% (more than 2X) less than it. In some aspects, the full dose is equal to 50% of the full dose corresponding to the next lowest dose group or is more than 50% less than it. In some aspects, the full dose is equal to 33% of the full dose corresponding to the next lowest dose group or is more than 33% less than it.

[0025] In some aspects, the methods further include the step of premedicating the patient with an agent that reduces the risk or severity of hypersensitivity before administering TNB-486. In some aspects, the agent that reduces the risk or severity of hypersensitivity is selected from the group consisting of dexamethasone, diphenhydramine, acetaminophen, ranitidine, tocilizumab, any equivalents thereof, or any combination thereof. In some aspects, the agent that reduces the risk or severity of hypersensitivity is administered from 15 minutes to 60 minutes before administering TNB-486. In some aspects, a 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.

[0026] In some aspects of the method, TNB-486 is administered in combination with another chemotherapy. In some aspects, the other chemotherapy is a combination of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunomycin), vincristine sulfate (Oncovin), and prednisone (R-CHOP). In some aspects, R-CHOP is administered on day 1 of the first treatment cycle and then on day 1 of each subsequent treatment cycle. In some aspects, each treatment cycle is 21 days long, R-CHOP is administered on day 1 of the first treatment cycle, the first priming dose is administered on day 8 of the first treatment cycle, the 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 aspects, R-CHOP and a therapeutically effective amount of TNB-486 are administered on day 1 of each subsequent treatment cycle.

[0027] In some aspects, patients have reduced cytokine release compared to different TNB-486 dosing regimens. In some aspects, patients do not experience cytokine release syndrome or only experience grade 1 cytokine release syndrome. In some aspects, cytokine release syndrome includes the release of IL-6 and / or TNF-α. In some aspects, the objective response rate (ORR) is 80% or higher. In some aspects, the complete response (CR) rate is 90% or higher. In some aspects, the 6-month PFS rate is 90% or higher. The method according to any one of the preceding claims, wherein TNB-486 is a bispecific molecule that binds to CD3 and human CD19, the bispecific molecule comprising: (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 portion that binds to human CD3; and (iii) a third polypeptide subunit that binds to human CD19 and comprises the amino acid sequence of SEQ ID NO:20.

[0028] These and other aspects will be further explained in the remainder of the present disclosure, including the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A is a schematic diagram showing an exemplary treatment method including a dose escalation phase (Group A) and two dose expansion phases (Group B and Group C). Figure 1B is a schematic diagram showing an exemplary treatment method including the administration of a combination of TNB-486 and R-CHOP (Group D).

[0030] Figure 2 is a schematic diagram showing an exemplary treatment method including a dose escalation phase (Group A), and providing additional details regarding the treatment of different patient groups.

[0031] Figure 3 It is a schematic diagram showing the recommended treatment guidelines for subjects presenting with signs and / or symptoms of cytokine release syndrome (CRS).

[0032] Figure 4 It is a schematic diagram of TNB-486, which is a trimeric antibody-like molecule (TCA) that contains one arm with a heavy chain / light chain pair that binds to CD3 and a second arm with only the heavy chain variable region that binds to CD19, in a monovalent configuration.

[0033] Figure 5A It is a graph of the percentage incidence of cytokine release syndrome at different administration time points as described in the examples. C1D1 - Cycle 1, Day 1. C1D15 - Cycle 1, Day 15. C2D1 - Cycle 2, Day 1. The total percentage of occurrences of CRS is indicated above each bar.

[0034] Figure 5B It is a graph of the percentage incidence of immune effector cell-associated neurotoxicity syndrome (ICANS) at different administration time points as described in the examples. C1D1 - Cycle 1, Day 1. C1D15 - Cycle 1, Day 15. C2D1 - Cycle 2, Day 1. The total percentage of occurrences of ICANS is indicated above each bar.

[0035] Figure 6 It is a schematic diagram of the patient outcomes of subjects evaluated at the first clinical cutoff as described in the examples. The lymphoma types are as shown in the legend. CR - Complete response. DC - PD - Discontinuation, patient decision. DC - Other - Discontinuation, other reasons. PR - Partial response.

[0036] Figure 7 It is a graph of the response rates at the first clinical cutoff as described in the examples. The first graph is for all types of B-cell non-Hodgkin lymphoma (B-NHL) subjects (n = 25). The second graph is for 4 subjects with diffuse large B-cell lymphoma (DLBCL). The third graph is for 8 subjects with follicular lymphoma (FL). The overall response rate (ORR) is shown at the top of the first and second graphs, and the complete response (CR) rate is shown in the third graph.

[0037] Figure 8 It is a schematic diagram of the patient outcomes of subjects evaluated at the second clinical cutoff as described in the examples. The lymphoma types are as shown in the legend. CR - Complete response. DC - PD - Discontinuation, patient decision. DC - Other - Discontinuation, other reasons. PR - Partial response.

[0038] Figure 9Shows the median cytokine levels (pg / ml) of IL-6 and TNF-α for various priming dose regimens.

[0039] Figure 10 Shows that complete response (CR) is usually achieved at the first assessment (week 8), and the 6-month PFS rate is 91%. Detailed implementation

[0040] Unless otherwise specified, the implementation of the methods of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are all within the scope of the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987 and updated regularly); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 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).

[0041] Where a range of values is provided, it is to be understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range (to one-tenth of the unit of the lower limit) and any other stated value or intervening value in the stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed within the disclosure, subject to any specific exclusions stated within the range. Where the range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0042] 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)).

[0043] 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 of ordinary skill in the art that the methods of the invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures known to those of ordinary skill in the art have not been described to avoid obscuring the methods disclosed herein.

[0044] All references cited throughout this disclosure (including patent applications and publications) are hereby incorporated by reference in their entirety.

[0045] I. Definitions

[0046] "Comprising" means that the recited element is essential in the composition / method / kits, but other elements may be included within the scope of the claims to form the composition / method / kits, etc.

[0047] "Consisting essentially of" means that the scope of the described composition or method is limited to the specified materials or steps that do not materially affect the basic and novel features of the subject method.

[0048] "Consisting of" means excluding any element, step, or ingredient not specified in the claims from the composition, method, or kit.

[0049] The antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. When referring to residues in the variable domain (about residues 1 - 113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al., ibid.). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody. Unless otherwise specified herein, referring to the residue numbering in the antibody variable domain means the residue numbering by the Kabat numbering system. Unless otherwise specified herein, referring to the residue numbering in the antibody constant domain means the residue numbering by the EU numbering system.

[0050] Antibodies (also referred to as immunoglobulins) generally comprise at least one heavy chain and one light chain, wherein the amino-terminal domains of the heavy and light chains are variable in sequence and are thus commonly referred to as variable domain, or variable heavy (VH) or variable light (VL) domains. The two domains generally associate to form a specific binding region, although as will be discussed herein, specific binding can also be obtained with only the heavy chain variable sequence, and a variety of non-native configurations of antibodies are known and used in the art.

[0051] A "functional" or "bioactive" antibody or antigen-binding molecule (including the single-chain antibodies and multispecific (e.g., bispecific) trimeric antibody-like molecules (TCA) described herein) is an antibody or antigen-binding molecule capable of exerting one or more of its natural activities in a structural, regulatory, biochemical, or biophysical event. For example, a functional antibody or other binding molecule (e.g., TCA) can have the ability to specifically bind an antigen, and this binding can in turn initiate or alter cellular or molecular events such as signal transduction or enzyme activity. A functional antibody or other binding molecule (e.g., TCA) can also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule (e.g., TCA) to exert one or more of its natural activities depends on several factors, including the correct folding and assembly of the polypeptide chains.

[0052] The term "adverse event" or "AE" is defined as any untoward medical event in a subject in a clinical study in which a pharmaceutical product is administered, and which does not necessarily have a causal relationship with the treatment. Thus, an AE can be any adverse and unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of the drug (investigational) product, whether or not the event is considered causally related to the use of the product.

[0053] 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, trispecific antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided that they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854 - 4861). Antibodies can be murine, human, humanized, chimeric, or from other species.

[0054] The term "antibody" can refer to a full-length heavy chain, full-length light chain, intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that contains an antigen-binding site that immunospecifically binds to a target of interest or a portion thereof, such targets including but not limited to cancer cells or cells that produce autoantibodies 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 altered Fc portions that provide reduced or enhanced effector cell activity. The light chain of the subject antibody can be a kappa light chain (Vκ) or a lambda light chain (Vλ). The immunoglobulins can be from any species. In one aspect, the immunoglobulins are predominantly human.

[0055] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up 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 an antigen. Monoclonal antibodies according to the methods of the invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be prepared by, for example, recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567).

[0056] The term "variable," when used in reference to an antibody, refers to certain portions of the antibody variable domains that are extensively different in sequence among antibodies and are used for binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, which mainly adopt a β-sheet conformation and are connected by three hypervariable regions, which form loops connecting the β-sheet structures and in some cases form parts of the β-sheet structures. The hypervariable regions in each chain are held tightly together by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody (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 do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as the antibody's involvement in antibody-dependent cell cytotoxicity (ADCC).

[0057] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the "complementary 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 those residues from the "hypervariable loops" residues 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" means the complementary determining regions of an antibody as defined in Lefranc, M.P. 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 defined herein.

[0058] This document presents exemplary CDR names. However, those skilled in the art will understand that many definitions of CDRs are commonly used, including the Kabat definition (see "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694 - 700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the positions 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 disclosed by: 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 hereby expressly incorporated by reference.

[0059] The terms "heavy-chain only antibody" and "heavy-chain antibody" are used interchangeably herein and, in the broadest sense, refer to an antibody or one or more portions of an antibody that lacks the light chain of a conventional antibody, such as one or more arms of an antibody. This term specifically includes, but is not limited to, homodimeric antibodies that contain a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs that are homodimers containing one variable domain (V-NAR) and five C-like constant domains (C-NAR), and functional fragments thereof; and soluble single-domain antibodies (sUniDabs TM ). In one aspect, a heavy-chain only antibody consists of a variable region antigen-binding domain that is composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another aspect, a heavy-chain only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another aspect, a heavy-chain only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In another aspect, a heavy-chain only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Also included herein are heavy-chain only antibodies in which the CH2 and / or CH3 domains are truncated. In another aspect, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but no hinge region. Heavy-chain only antibodies can be in the form of a dimer in which the two heavy chains are disulfide-bonded or otherwise covalently or non-covalently linked to each other. Heavy-chain only antibodies can belong to the IgG subclass, but antibodies belonging to other subclasses (such as IgM, IgA, IgD, and IgE subclasses) are also included herein. In a particular aspect, the heavy-chain antibody is an IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one aspect, 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 TM ), called UniAbs TM . The variable region (VH) of UniAbs TM is called UniDabs TM and is a universal structural unit that can be linked to an Fc region or serum albumin for the development of novel therapeutic agents with multispecificity, increased potency, and extended half-life. Since homodimeric UniAbs TM lack a light chain and thus lack a VL domain, an antigen is recognized by a single domain, namely the variable domain of the heavy chain of a heavy-chain antibody (VH or VHH).

[0060] As used herein, a "complete antibody chain" is an antibody chain that comprises a full-length variable region and a full-length constant region (Fc). A complete "conventional" antibody comprises a complete light chain and a complete heavy chain, as well as the light chain constant domain (CL) and the heavy chain constant domains CH1, hinge, CH2, and CH3 of secreted IgG. Other isotypes (such as IgM or IgA) may have different CH domains. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. A complete antibody may have one or more "effector functions", which refers to those biological activities that can be attributed to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter effector characteristics, binding to Fc receptors, etc.

[0061] Antibodies and various antigen-binding proteins can be provided as different classes based on the amino acid sequence of the Fc (constant domain) of their heavy chains. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hinge-less forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types based on the amino acid sequence of their constant domains, called κ (kappa) and λ (lambda). Antibodies according to aspects of the methods may comprise a κ light chain sequence or a λ light chain sequence.

[0062] "Functional Fc region" has the "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require interaction of the Fc region with receptors such as FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and the low affinity FcRn receptor; and can be evaluated using a variety of assays known in the art. "Dead" or "silent" Fc is an Fc that has been mutated to retain activity with respect to, for example, extended serum half-life, but does not activate high affinity Fc receptors, or has a reduced affinity for Fc receptors.

[0063] "Native sequence Fc region" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0064] "Variant Fc region" contains an amino acid sequence that differs from a native sequence Fc region due to 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 the native sequence Fc region or the Fc region of a parental polypeptide, e.g., from about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parental polypeptide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, and preferably from about one to about five amino acid substitutions. Variant Fc regions herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parental polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0065] The human IgG4 Fc amino acid sequence (UniProtKB No. P01861) is incorporated herein. Silent IgG1 is described, for example, in Boesch, A.W. et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4): p. 915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0066] Other Fc variants are possible, including but not limited to variants in which regions capable of forming disulfide bonds are deleted, or in which certain amino acid residues at the N-terminus of the native Fc are eliminated, or methionine residues are added thereto. Thus, in some aspects, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another aspect, the hinge region of the Fc may be completely removed. In yet another aspect, the antibody may comprise an Fc variant.

[0067] In addition, Fc variants can be constructed to achieve complement binding or Fc receptor binding by substituting (mutating), deleting or adding amino acid residues, thereby removing or significantly reducing effector function. For example, but not limited to, deletions can 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 Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, prenylation, acetylation, amidation, etc.

[0068] In some aspects, the antibody comprises a variant human IgG4 CH3 domain sequence containing a T366W mutation, which may optionally be referred to herein as the IgG4 CH3 "stalk" sequence. In some aspects, the antibody comprises a variant human IgG4 CH3 domain sequence containing T366S, L368A, and Y407V mutations, which may optionally be referred to herein as the IgG4 CH3 "socket" sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner so as to place the "stalk" on the first heavy chain constant region of the first monomer in the antibody dimer, and the "socket" on the second heavy chain constant region of the second monomer in the antibody dimer, thereby facilitating the correct pairing (heterodimerization) of the desired heavy chain polypeptide subunits in the antibody.

[0069] In some aspects, the antibody comprises a heavy chain polypeptide subunit containing a variant human IgG4 Fc region comprising S228P, F234A, L235A, and T366W (stalk) mutations. In some aspects, the antibody comprises a heavy chain polypeptide subunit containing a variant human IgG4 Fc region comprising S228P, F234A, L235A, T366S, L368A, and Y407V (socket) mutations.

[0070] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, an antibody having an Fc region according to the present disclosure can comprise an antibody with or without K447.

[0071] Aspects of the present disclosure include antibodies comprising only heavy chain variable regions in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" as applied to a heavy chain variable region domain means that only one heavy chain variable region domain is present, which has a single binding site. In contrast, the term "bivalent configuration" as applied to a heavy chain variable region domain means that two heavy chain variable region domains (each having a single binding site) are present and are linked by a linker sequence (see Figure 4 ). Non-limiting examples of linker sequences are further discussed herein and include, but are not limited to, GS linker sequences of various lengths. When the heavy chain variable regions are in a bivalent configuration, each of the two heavy chain 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 otherwise specifically indicated, heavy chain variable regions designated as "bivalent configuration" should be understood to contain two identical heavy chain variable region domains linked by a linker sequence, wherein each of the two identical heavy chain variable region domains has binding affinity for the same target antigen.

[0072] Aspects of the present disclosure include antibodies having a multispecific configuration, including but not limited to bispecific, trispecific, etc. A variety of methods and protein configurations are known and are used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0073] A variety of methods for generating multivalent artificial antibodies have been developed by recombinant fusion of the variable domains of two or more antibodies. In some aspects, a first antigen domain and a second antigen-binding domain on a polypeptide are linked by a polypeptide linker. A non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by a serine residue, and wherein this sequence is repeated n times, where n is an integer 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 and are described, for example, 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.

[0074] The term "trichain 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, wherein two comprise, consist essentially of, or consist of a heavy chain and a light chain of a monoclonal antibody or a functional antigen-binding fragment of such antibody chains, comprising an antigen-binding region and at least one CH domain. The heavy chain / 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 that comprises an Fc portion that comprises CH2 and / or CH3 and / or CH4 domains 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 the variable region of an antibody heavy chain or light chain or have sequence identity with the variable region of an antibody heavy chain or light chain. Portions of such variable regions may be encoded by V H and / or V L gene segments, D and J H gene segments, or J L gene segments. The variable region may be encoded by rearranged V H DJ H , V L DJ H , V H J L or V L J L gene segments.

[0075] The TCA-binding compound utilizes "heavy-chain only antibody" or "heavy-chain antibody" or "heavy-chain polypeptide", as used herein, which means a single-chain antibody comprising the heavy-chain constant regions CH2 and / or CH3 and / or CH4 but lacking the CH1 domain. In one aspect, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 and CH3 domains. In another aspect, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH2 domain. In another aspect, the heavy-chain antibody consists of an antigen-binding domain, at least a portion of the hinge region, and the CH3 domain. Also included herein are heavy-chain antibodies in which the CH2 and / or CH3 domains are truncated. In another aspect, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain but without a hinge region. The heavy-chain only antibody can be in the form of a dimer, in which the two heavy chains are disulfide-bonded or otherwise covalently or non-covalently linked to each other, and can optionally include an asymmetric interface between one or more CH domains to facilitate proper pairing between the polypeptide chains. The heavy-chain antibody can 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 a particular aspect, the heavy-chain antibody is an IgG1, IgG2, IgG3 or IgG4 subtype, especially the IgG1 subtype or the IgG4 subtype. Non-limiting examples of the TCA-binding compound are described, for example, in WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0076] Heavy chain antibodies constitute approximately one quarter of the IgG antibodies produced by Camelidae animals (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. Thus, the variable antigen-binding part is called the VHH domain, and it represents the smallest naturally occurring complete antigen-binding site, with a length of only about 120 amino acids (Desmyter, A. et al. J. Biol. Chem. 276, 26285 - 26290 (2001)). Heavy chain antibodies with high specificity and affinity against various antigens can be generated by immunization (vander Linden, R.H. et al. Biochim. Biophys. Acta. 1431, 37 - 46 (1999)), and the VHH part can be easily cloned and expressed in yeast (Frenken, L.G.J. et al. J. Biotechnol. 78, 11 - 21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M.A. et al. FEBS Lett. 414, 521 - 526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, 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)).

[0077] As used herein, the terms "CD19" and "cluster of differentiation 19" refer to a molecule expressed during all stages of B cell development up to terminal differentiation into plasma cells. The term "CD19" includes CD19 proteins of any human and non-human animal species, and specifically includes human CD19 as well as CD19 of non-human mammals.

[0078] As used herein, the term "human CD19" includes any variant, isotype, and species homolog of human CD19 (UniProt P15391), regardless of its source or method of preparation. Thus, "human CD19" includes human CD19 naturally expressed by cells and CD19 expressed on cells transfected with the human CD19 gene.

[0079] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex consists of six different polypeptide chains. These include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt 20963), and it is associated with the T cell receptor α and β chains. Unless otherwise specified, the term "CD3" includes any CD3 variants, isotypes, and species homologs that are naturally expressed by cells (including T cells) or that can be expressed on cells transfected with the genes or cDNAs encoding those polypeptides.

[0080] A "CD19×CD3 antibody" is a multispecific single-chain antibody, such as a bispecific single-chain antibody, that comprises two different antigen-binding regions, one of which specifically binds to CD19 and one of which specifically binds to CD3.

[0081] The terms "anti-CD19 single-chain antibody only", "CD19 single-chain antibody only", "anti-CD19 single-chain antibody", and "CD19 single-chain antibody" are used interchangeably herein to refer to a single-chain antibody as defined above that immunospecifically binds to CD19 as defined above, including human CD19 as defined above. This definition includes, but is not limited to, human single-chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulins, including UniRatslM that produces the human anti-CD19 UniAblM antibody as defined above.

[0082] The "percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this document, the percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

[0083] A "separated" antibody is an antibody that has been identified and separated and / or recovered from the components of its natural environment. The contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and can include enzymes, hormones, and other proteins or non-proteinaceous solutes. In a preferred aspect, the antibody is purified (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminus or internal amino acids by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Separated antibodies include antibodies in situ within recombinant cells because at least one component of the natural environment of the antibody will be absent. However, generally, separated antibodies will be prepared by at least one purification step.

[0084] The 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, such as higher-order multi-epitope specificities, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody", "multispecific single-chain antibody", "multispecific heavy-chain antibody", "multispecific UniAb" TM ", and "multispecific binding compound" are used herein in the broadest sense and encompass all antibodies having more than one binding specificity. The multispecific heavy-chain anti-CD19 antibodies of the present disclosure specifically include antibodies that immunospecifically bind to a single epitope on the CD19 protein (such as human CD19) and an epitope on a different protein (such as, for example, the CD3 protein) (i.e., bivalent and monocomplements). The multispecific heavy-chain anti-CD19 antibodies of the present disclosure specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on the CD19 protein (such as human CD19) (i.e., bivalent and bicep). The multispecific heavy-chain anti-CD19 antibodies of the present disclosure also specifically include antibodies that immunospecifically bind to an epitope on the CD19 protein (such as human CD19) and an epitope on a different protein (such as, for example, the CD3 protein, such as human CD3) (i.e., bivalent and bicep). The 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 the CD19 protein (such as human CD19 protein) and an epitope on a different protein (such as, for example, the CD3 protein, such as human CD3 protein) (i.e., trivalent and bicep).

[0085] The antibodies of the present disclosure include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, and antibodies comprising more than one binding unit having the same binding specificity. The terms "monospecific antibody", "monospecific only heavy chain antibody", "monospecific heavy chain antibody" and "monospecific UniAb" TM " are used herein in the broadest sense and encompass all antibodies having one binding specificity. The monospecific heavy chain anti-CD19 antibodies of the present disclosure specifically include antibodies that immunospecifically bind to an epitope (monovalent and monospecific) on the CD19 protein, such as human CD19. The monospecific heavy chain anti-CD19 antibodies of the present disclosure also specifically include antibodies having more than one binding unit that immunospecifically bind to an epitope on the CD19 protein, such as human CD19 (e.g., multivalent antibodies). For example, the monospecific antibodies according to aspects of the present disclosure may include a heavy chain variable region comprising two antigen-binding domains, wherein each antigen-binding domain binds to the same epitope on the CD19 protein (i.e., bivalent and monospecific).

[0086] An "epitope" is the 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 reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0087] "Epitope mapping" is the process of identifying the binding site or epitope of an antibody on its target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous amino acid sequence in a protein. Conformational epitopes are formed by amino acids that are not contiguous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0088] As used herein, the term "valence" refers to the specified number of binding sites in an antibody molecule.

[0089] A "monovalent" antibody has one binding site. Thus, a monovalent antibody is also monospecific.

[0090] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent", "trivalent" and "tetravalent" refer to the presence of two, three and four binding sites, respectively. Thus, bispecific antibodies according to the present disclosure are at least bivalent and can be trivalent, tetravalent or other multivalent. Bivalent antibodies according to aspects of the present disclosure can have two binding sites directed against the same epitope (i.e., bivalent, monocompetitive) or against two different epitopes (i.e., bivalent, biconcompetitive).

[0091] A variety of methods and protein configurations are known and are used to prepare bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0092] The term "trimeric 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, wherein two comprise, consist essentially of, or consist of a heavy chain and a light chain of a monoclonal antibody or a functional antigen-binding fragment of such antibody chains, comprising an antigen-binding region and at least one CH domain. The heavy chain / 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 that comprises 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 the variable region of an antibody heavy or light chain or has sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions may be encoded by V H and / or V L gene segments, D and J H gene segments or J L gene segments. The variable region may be encoded by rearranged V H DJ H 、V L DJ H 、V H J L or V L J L gene segments. The TCA protein utilizes a heavy chain-only antibody as defined above.

[0093] As used herein, the term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced in vivo by random or site-specific mutagenesis or in vivo by somatic hypermutation. The term "human antibody" specifically includes heavy chain-only antibodies having human heavy chain variable region sequences produced by transgenic animals (such as transgenic rats or mice), particularly UniAbs TM produced by UniRats TM , as defined above.

[0094] A "chimeric antibody" or "chimeric immunoglobulin" means an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, such as a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies having a non-human or artificial Fc region, as well as human allotypes. Such immunoglobulins can be isolated from the animals of the present disclosure that have been engineered to produce such chimeric antibodies.

[0095] As used herein, the term "effector cell" refers to immune cells that participate in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some aspects, effector cells such as natural killer cells are capable of inducing antibody-dependent cell cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens. In some aspects, effector cells can phagocytose target antigens or target cells.

[0096] A "human effector cell" is a white blood cell that expresses receptors such as the T cell receptor or FcR and performs effector functions. Preferably, the cell expresses at least FcγRIII and performs an ADCC effector function. Examples of human white blood cells that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among which NK cells are preferred. Effector cells can be isolated from their natural sources, such as from blood or PBMC as described herein.

[0097] 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 cytotoxic T cells (CTL)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear leukocytes such as neutrophils, granulocytes, mast cells, and basophils.

[0098] Antibody "effector functions" refer to those biological activities that can be attributed to the Fc region of an antibody (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0099] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. The major cell mediating ADCC, the NK cell, expresses only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) summarizes FcR expression on hematopoietic cells. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed by Clynes et al., PNAS (USA) 95:652-656 (1998).

[0100] "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 can be performed, for example, as described by Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0101] 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 transformation), mantle cell lymphoma (MCL), follicular lymphoma (FL, grades 1-3), or marginal zone lymphoma (MZL).

[0102] "CD19-positive" is or refers to the expression of CD19 on ≥50% of tumor cells, as evaluated by a pathologist using immunohistochemistry (IHC) or flow cytometry.

[0103] The term "characterized by CD19 expression" broadly refers to any disease or disorder in which CD19 expression is associated or related to one or more pathological processes that are characteristic of the disease or disorder. Such disorders include, but are not limited to, B-cell tumors.

[0104] "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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigen more rapidly and tend to remain bound.

[0105] As used herein, "Kd" or "Kd value" refers to the dissociation constant determined in kinetic mode by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA). For example, an anti-mouse Fc sensor is loaded with mouse-Fc fusion antigen and then immersed in a well containing an antibody to measure the concentration-dependent association rate (kon). In the final step, the antibody dissociation rate (koff) is measured, where the sensor is immersed in a well containing only buffer. Kd is the ratio of koff / kon. (See further details in Concepcion, J et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0106] The term "treatment" and the like are used herein to generally mean obtaining the desired pharmacological and / or physiological effect. This effect can be prophylactic, i.e., preventing a disease or its symptoms completely or in part, and / or can be therapeutic, i.e., curing a disease and / or adverse effects attributable to the disease partially or completely. As used herein, "treatment" encompasses any treatment of a disease in a mammal and includes: (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing its progression; or (c) alleviating the disease, i.e., causing the disease to regress. A therapeutic agent can be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. Such treatment is ideally carried out before the complete loss of function of the affected tissue. The subject therapy can be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0107] "Therapeutically effective amount" means the amount of an active agent necessary to confer a therapeutic benefit on a subject. For example, a "therapeutically effective amount" is an amount that induces, improves, or otherwise causes an improvement in the pathological symptoms, disease progression, or physiological condition associated with a disease or an improvement in resistance to a disorder.

[0108] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one aspect, the mammal is a human. The terms "subject", "individual", and "patient" encompass, but are not limited to, individuals suffering from cancer, autoimmune diseases, pathogen infections, etc. The subject can be a human, but also includes other mammals, particularly those that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0109] The term "pharmaceutical preparation" refers to a preparation in a form such that the biological activity of the active ingredient is effective and that does not contain additional components that are unacceptable toxic to the subject to whom the preparation will be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can be reasonably administered to the test mammal to provide an effective dose of the active ingredient employed.

[0110] A "sterile" preparation is sterile or free or substantially free of all live microorganisms and their spores. A "frozen" preparation is a preparation at a temperature below 0°C.

[0111] A "stable" formulation is one in which the protein substantially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability as well as its biological activity upon storage. The storage period is typically selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247 - 301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29 - 90 (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively evaluated in a number of different ways, including assessment of aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessment of charge heterogeneity by using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessment of the biological activity or antigen-binding function of the antibody; 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), cleavage / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0112] Abbreviations used in this text 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 (fluorescent in situ hybridization); FL (follicular lymphoma); GCP (good clinical practice); G-CSF (granulocyte colony-stimulating factor); GLP (good laboratory practice); 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's brochure); ICE (immune effector cell encephalopathy); ICF (informed consent form); ICH (International Council for Harmonisation); IEC (independent ethics committee); IMT (immune-mediated toxicity); IRB (institutional review board); IV (intravenous); mAb (monoclonal antibody); MABEL (minimum anticipated biological effect level); MED (minimum effective dose); MedDRA (Medical Dictionary for Regulatory Activities); MR (minor response); MRI (magnetic resonance imaging); MTD (maximum tolerated dose); NA (not applicable); NCA (non-compartmental model analysis); NCCN (National Comprehensive Cancer Network); NCI (National Cancer Institute); B-NHL (B-cell non-Hodgkin lymphoma); NLCB (no longer clinical benefit); NT (neurotoxicity); ORR (objective response rate); OS (overall survival); PARPi (poly-ADP-ribose-polymerase inhibitor); PBMC (peripheral blood mononuclear cell); PC (positive control); PD (pharmacodynamics or progressive disease); PET (positron emission tomography); PI (prescribing information); PK (pharmacokinetics); PFS (progression-free survival); PO (per os, orally); PR (partial response); PT (prothrombin time);Q3W (once every three weeks); QTc (QT interval corrected for heart rate); RLT (radio-ligand therapy); rPFS (radiographic progression-free survival); RP2D (recommended phase 2 dose); RR (relapsed or refractory); SAE (serious adverse event); SCT (stem cell transplantation); 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-BsAbs (T cell-engaging bispecific antibodies); TEAE (treatment-emergent adverse events); TLS (tumor lysis syndrome); Tmax (time to reach maximum observed serum concentration); TTP (time to progression); TTR (time to response); ULN (upper limit of normal); US (United States); V1 (volume of the central compartment); Vss (volume of distribution at steady state); WHO (World Health Organization).

[0113] II. TNB-486 (anti-CD3 / anti-CD19)

[0114] The present disclosure relates to methods of treating non-Hodgkin lymphoma by administering to a patient in need thereof a bispecific trimeric antibody-like molecule (TCA). In some aspects, the TCA is designated TNB-486 and comprises an anti-CD3 binding domain paired with an anti-CD19 VH binding domain as shown in Figure 4 wherein the anti-CD3 VH domain and the anti-CD3 VL domain together have a binding affinity for CD3, and the TCA further comprises a heavy chain variable domain of a single-chain antibody having a binding affinity for CD19 in a monovalent configuration, and the TCA further comprises a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence containing S228P, F234A, L235A, and T366W mutations (pestle), and a second heavy chain constant region sequence containing S228P, F234A, L235A, T366S, L368A, and Y407V mutations (mortar).

[0115] In some aspects, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain aspects, the light chain is a fixed light chain. In some aspects, 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 within a human VH framework. In some aspects, 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 within a human VL framework. The CD3-binding VH domain and the light chain variable domain together have binding affinity for CD3. In some aspects, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 7. In some aspects, the CD3-binding VH domain comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the heavy chain variable region sequence of SEQ ID NO: 7. In some aspects, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 8. In some aspects, the fixed light chain comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the heavy chain variable region sequence of SEQ ID NO: 8.

[0116] Multispecific antibodies comprising the above-described CD3-binding VH domain and light chain variable domain have advantageous properties, for example, as described in PCT Publication No. WO2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety.

[0117] Table 1. Amino Acid Sequences of Anti-CD3 Heavy and Light Chain CDR1, CDR2, CDR3 .

[0118]

[0119] Table 2. Amino Acid Sequences of Anti-CD3 Heavy and Light Chain Variable Regions .

[0120]

[0121] Table 3: Human IgG4 Fc Region Sequence with Silent Mutations .

[0122]

[0123] Table 4: Additional Sequences .

[0124]

[0125]

[0126]

[0127]

[0128] In some aspects, bispecific or multispecific antibodies are provided, which can have any of the configurations discussed herein, including but not limited to bispecific trispecific antibody-like molecules. In some aspects, a bispecific antibody can comprise at least one heavy chain variable region having binding specificity for CD19, and at least one heavy chain variable region having binding specificity for a different protein (e.g., CD3). In some aspects, a bispecific antibody can comprise a heavy chain / light chain pair having binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody, which comprises an Fc portion that comprises 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 in a monovalent or bivalent configuration. In a particular aspect, the bispecific antibody comprises a heavy chain / light chain pair having binding specificity for an antigen on an effector cell (e.g., the CD3 protein on a T cell) in a monovalent or bivalent configuration, and a heavy chain from a heavy chain-only antibody that comprises an antigen-binding domain having binding specificity for CD19.

[0129] In some aspects, where the antibodies of the present disclosure are bispecific antibodies, one arm (a binding moiety or a binding unit) of the antibody is specific for human CD19, while the other arm can be specific for a target cell, a tumor-associated antigen, a targeting antigen (such as integrin, etc.), a pathogen antigen, a checkpoint protein, etc. The target cells specifically include cancer cells. In some aspects, one arm (a binding moiety or a binding unit) of the antibody is specific for human CD19, while the other arm is specific for CD3.

[0130] In a preferred aspect, the antibody is a bispecific TCA, which comprises a first polypeptide containing SEQ ID NO:11, a second polypeptide containing SEQ ID NO:18, and a third polypeptide containing SEQ ID NO:20. In some aspects, CDR1 comprises a polypeptide containing SEQ ID NO:21. In some aspects, CDR2 comprises a polypeptide containing SEQ ID NO:22. In some aspects, CDR3 comprises a polypeptide containing SEQ ID NO:23. The antibody is also referred to as TNB-486 and is also described in U.S. Patent No. 11,390,681 B2 and PCT Publication WO2021 / 222578, the disclosures of which are incorporated herein by reference in their entireties.

[0131] III. Preparation of Antibodies

[0132] The multispecific antibodies of the present disclosure can be prepared by methods known in the art. In a preferred aspect, the heavy chain antibodies herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which the endogenous immunoglobulin genes are knocked out or inactivated. In a preferred aspect, the heavy chain antibodies herein are produced in UniRat TM . UniRat TM silences its endogenous immunoglobulin genes and uses the human immunoglobulin heavy chain transgenic locus to express a diverse, naturally optimized repertoire of fully human HCAb. Although a variety of techniques can be used to knock out or silence the endogenous immunoglobulin locus in rats, in UniRat TM , zinc finger (endonuclease) (ZNF) technology is used to inactivate the endogenous rat heavy chain J locus, the light chain Cκ locus, and the light chain Cλ locus. The ZNF constructs for microinjection into oocytes can generate IgH and IgL knockout (KO) lines. See, for example, Geurts et al., 2009, Science 325:433 for details. Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941 reported the characterization of Ig heavy chain knockout rats. The advantage of ZNF technology is that non-homologous end joining by deletion of up to several kb to silence a gene or locus can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). The human heavy chain antibodies produced in UniRat TM are called UniAbs TM , and can bind epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity, and small size make them ideal for monospecific and multispecific applications.

[0133] In addition to UniAbs TM , specifically included herein are heavy chain-only antibodies lacking the camelid VHH framework and mutations and their functional VH regions. Such heavy chain-only antibodies can be produced, for example, in transgenic rats or mice that contain a fully human heavy chain-only locus as described, for example, in WO2006 / 008548, but other transgenic mammals such as rabbits, guinea pigs, rats, rats and mice are preferred. Heavy chain-only antibodies (including their VHH or VH functional fragments) can also be produced by recombinant DNA technology by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (such as CHO cells), Escherichia coli, or yeast.

[0134] The domains of the heavy-chain-only antibodies combine the advantages of antibodies and small molecule drugs: they can be monovalent or multivalent; they have low toxicity; and they are inexpensive to manufacture. 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 adjusted according to the desired use or indication. In addition, the VH and VHH domains of HCAb can be manufactured in a cost-effective manner.

[0135] In one particular aspect, the heavy-chain antibodies of the present disclosure (including UniAbs TM ) have a native amino acid residue at the first position in the FR4 region (amino acid position 101 according to the Kabat numbering system), which is replaced by another amino acid residue that is capable of disrupting a surface-exposed hydrophobic patch that includes the native amino acid residue at that position or is associated with the native amino acid residue at that position. Such hydrophobic patches are typically buried at the interface with the constant region of the antibody light chain, but become surface-exposed in HCAb and are at least partially responsible for the unwanted aggregation and light chain association of HCAb. The substituted amino acid residue is preferably charged, and more preferably positively charged, such as lysine (Lys, K), arginine (Arg, R), or histidine (His, H), preferably arginine (R). In a preferred aspect, the heavy-chain-only antibodies derived from transgenic animals contain a Trp to Arg mutation at position 101. The resulting HCAb preferably has high antigen-binding affinity and solubility in the absence of aggregation under physiological conditions.

[0136] As part of the present disclosure, a human IgG anti-CD19 heavy-chain antibody with a unique sequence from UniRatlM animals (UniAblM) was identified in an ELISA (recombinant CD19 extracellular domain) protein and cell-binding assay that binds human CD19. The identified heavy-chain variable region (VH) sequence (see Figure 2 ) is positive for human CD19 protein binding and / or for binding to CD19+ cells, and negative for binding to cells that do not express CD19.

[0137] The antibodies described herein bind to the CD19-positive Burkitt's lymphoma cell lines Daudi ( CCL-2131M), Raji ( CCL-86 TM ) and Ramos ( CRL-1596TM), and some cross-react with the CD19 protein of cynomolgus macaques. In addition, if desired, they can be engineered to provide cross-reactivity with the CD19 protein of any animal species.

[0138] Anti-CD19 heavy chain antibodies (such as the UniAbs of the present disclosure TM ) may have an affinity for CD19 with a Kd of from about 10 -6 to about 10 -11 , including but not limited to: from about 10 -6 to about 10 -10 ; from about 10 -6 to about 10 -9 ; from about 10 -6 to about 10 -8 ; from about 10 -8 to about 10- 11 ; from about 10 -8 to about 10 -10 ; from about 10 -8 to about 10 -9 ; from about 10 -9 to about 10 -11 ; from about 10 -9 to about 10 -10 ; or any value within these ranges. Affinity selection can be confirmed with a biological assessment that modulates (e.g., blocks) CD19 bioactivity, including in vitro assays, preclinical models, and clinical trials, as well as an assessment of potential toxicity.

[0139] Heavy chain antibodies that bind to non-overlapping epitopes on the CD19 protein (e.g., UniAbs TM ) can be identified by a competitive binding assay (such as an enzyme-linked immunosorbent assay (ELISA assay) or a flow cytometry competitive binding assay). For example, competition can be exploited between a known antibody that binds to the target antigen and the antibody of interest. By using this method, a group of antibodies can be divided into antibodies that compete with a reference antibody and antibodies that do not compete with the reference antibody. Non-competing antibodies are identified as binding to a unique epitope that does not overlap with the epitope bound by the reference antibody. Typically, one antibody is immobilized, the antigen is bound, and the ability of a second labeled (e.g., biotinylated) antibody to bind the captured antigen is tested in an ELISA assay. This can also be performed using a surface plasmon resonance (SPR) platform, including ProteOn XPR36 (BioRad, Inc.), Biacore2000, and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibistechnologies B.V.), as well as on a biolayer interferometry platform (such as Octet Red384 and Octet HTX (ForteBio, Pall Inc.)). Further details are provided in the Examples herein.

[0140] Typically, an antibody "competes" with a reference antibody if it causes a reduction of about 15% to 100% in binding of the reference antibody to the target antigen, as determined by standard techniques, such as by the competition binding assays described above. In various aspects, the relevant 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.

[0141] Drug Compositions

[0142] Another aspect of the present disclosure provides a pharmaceutical composition comprising one or more multispecific binding compounds of the present disclosure mixed with a suitable pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carriers are exemplary, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to accommodate therapeutic components, or combinations thereof.

[0143] In one aspect, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb) that binds to CD19. TM In another aspect, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb) having binding specificity for two or more non-overlapping epitopes on the CD19 protein. TM In a preferred aspect, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb) that has binding specificity for CD19 and a binding target on an effector cell (e.g., a binding target on a T cell, such as a CD3 protein on a T cell). TM ).

[0144] A pharmaceutical composition of an antibody for use according to the present disclosure is prepared for storage by mixing a protein having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences 16th edition, edited by Osol, A. (1980)), such as in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 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); nonionic surfactants, such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0145] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and are produced under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage forms (i.e., a dosage for a single administration). The formulation depends on the selected route of administration. 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 contain a carrier, excipient, or stabilizer as described above. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

[0146] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies of the present disclosure, including UniAbs TM . Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.

[0147] IV. Methods of Use

[0148] The antibodies and pharmaceutical compositions described herein can be used to treat diseases and disorders characterized by the expression of a target protein (e.g., CD3, CD19), including but not limited to the disorders and conditions further described herein. In a preferred aspect, the antibodies and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by CD19 expression.

[0149] The pharmaceutical compositions herein comprising anti-CD19 antibodies can be used to treat conditions characterized by CD19 expression, including but not limited to non-Hodgkin lymphoma.

[0150] The effective dose of the compositions of the present disclosure for treating a disease varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals can also be treated, such as companion animals, such as dogs, cats, horses, etc., laboratory mammals, such as rabbits, mice, rats, etc., and so on. The treatment dose can be titrated to optimize safety and efficacy.

[0151] Generally, the compositions are prepared to be injectable, as a liquid solution or suspension; solid forms suitable for dissolution or suspension in a liquid vehicle 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. As described above, the formulations can also be emulsified or encapsulated in liposomes or microparticles (such as poly(lactide), poly(glycolide), or copolymers) to enhance adjuvant effects. 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 long-acting injections or implantable formulations, which can be formulated in a manner that permits sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0152] The toxicity of the antibodies and antibody constructs described herein can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., by determining the LD50 (the dose lethal to 50% of the population) or LD100 (the dose lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic to humans. The dose of the antibodies described herein is preferably within a range of circulating concentrations, including effective doses with little or no toxicity. The dose can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dose can be selected by the individual physician according to the condition of the patient.

[0153] Compositions for administration will generally comprise 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 and the like. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable adjunct substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, etc., 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 according to the particular mode of administration chosen and the needs of the patient, based on fluid volume, viscosity, body weight, etc. (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gilman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0154] Kits containing the active agents of the present disclosure, their formulations, and instructions for use are also within the scope of the present disclosure. The kits can also include at least one additional reagent, such as a chemotherapeutic agent, etc. The kits generally include a label indicating the intended use of the kit contents. As used herein, the term "label" includes any written or recorded material on or supplied with the kit, or otherwise accompanying the kit.

[0155] Aspects of the present disclosure include methods for evaluating the safety, clinical pharmacokinetics (PK), and clinical activity of TNB-486 in subjects with B-NHL. As noted above, B-NHL subtypes can 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 the current 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).

[0156] In some aspects, the patient has received at least 2 lines of systemic therapy approved for B-NHL. In some aspects, the patient has been exposed to a typical second-line therapy, is intolerant to it, does not meet the criteria for it, or has refused it, such as high-dose chemotherapy followed by autologous (stem cell transplantation (SCT)).

[0157] Aspects of the present disclosure include methods related to single - agent dose escalation (Part 1, Group A) and single - agent dose expansion (Part 2, Groups B and C). The schematic of the study design for the administration is presented in Figure 1A . Some aspects include methods related to the combination of TNB - 486 dose escalation with R - CHOP (Part 3, Group D). The schematic of the administration for the combination dosing regimen is presented in Figure 1B .

[0158] Aspects of the present disclosure include administration methods in which dose - limiting toxicity (DLT) occurs at a lower rate than other administration methods. In some aspects, the DLT that occurs is cytokine release syndrome (CRS). CRS and its severity grades are described herein. In some aspects, patients do not experience CRS or only experience Grade 1 CRS. In some aspects, patients do not experience CRS. In some aspects, patients do not experience CRS or only experience Grade 1 or 2 CRS. In some aspects, any CRS experienced by the patient resolves without treating CRS. In some aspects, any CRS experienced by the patient resolves by treating CRS. In aspects, CRS is treated by administering tocilizumab.

[0159] In some aspects of the method, the chance that a patient experiences Grade 2 or 3 CRS is less than 20%. In aspects, the chance that a patient experiences Grade 2 or 3 CRS is less than 24%. In aspects, the chance that a patient does not experience CRS is 40%.

[0160] In aspects, patients experience CRS only during the first administration cycle. In aspects, the chance that a patient experiences CRS on Day 1 of the first administration cycle is about 53% or less. In aspects, the chance that a patient experiences CRS on Day 15 of the first administration cycle is about 15% or less. In aspects, patients do not experience CRS during the second administration cycle. In aspects, patients do not experience CRS during the second administration cycle or any subsequent administration cycle.

[0161] Single Therapy Dose Escalation (Part 1, Group A) :

[0162] In some aspects, the method involves evaluating the safety, tolerability, PK, and PD profiles of single - agent TNB - 486 therapy administered once every four weeks (Q2W, 28 - day cycle) in patients with B - NHL who have received at least 2 prior lines of therapy. In some aspects, the patients have been previously exposed to typical second - line therapies, are intolerant to them, do not meet the criteria for them, or have refused them, such as high - dose chemotherapy followed by autologous (stem cell transplantation (SCT)). In Group A, the cohort will initially enroll a single subject.

[0163] In some aspects, the method involves administering a single dose of TNB-486 over a 28-day cycle for at least one cycle. In some aspects, the TNB-486 doses are 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 aspects, the method involves varying the dosing frequency to include additional time between doses. In some aspects, the treatment cycles are repeated two or more times. In some aspects, TNB-486 is administered to a patient as a single therapy. In some aspects, TNB-486 is administered by intravenous infusion (IV).

[0164] In some aspects, a patient will receive one or more priming doses prior to the target dose. In some aspects, a patient will receive a single priming dose on day 1 of the first dosing cycle. In some aspects, a patient will receive two priming doses. In some aspects, a patient will receive two priming doses, where the first priming dose is administered on day 1 of the first dosing cycle and the second priming dose is administered on day 2 of the first dosing cycle.

[0165] In some aspects, Group A involves a dose-escalation design to evaluate the safety, tolerability, PK, and PD profiles of the single agent TNB-486 administered Q2W in up to 24 patients with B-NHL who have received at least 2 prior lines of therapy. In some aspects, the patients have been previously exposed to a treatment regimen known to provide clinical benefit in B-NHL, are intolerant to it, do not meet the criteria for it, have refused it, or are not suitable candidates for it. In Group A, the cohort initially enrolls a single subject. In some aspects, nine dose levels of TNB-486 (Table) were evaluated, but the number of dose levels tested depends on safety, PK / PD, and activity data.

[0166] In Group A, the cohort will initially enroll a single subject ( Figure 2 and Table 5). Eight dose levels (Table 5) were proposed for TNB-486, but the number of dose levels tested will depend on tolerability. In some aspects, the 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.

[0167] In aspects where at least one priming dose is administered, the priming dose is from 150 μg to 1500 μg. In various aspects, the priming dose is from 175 μg to 1250 μg. In various aspects, the priming dose is from 200 μg to 1000 μg. In various aspects, the priming dose is from 200 μg to 500 μg. In various aspects, the priming dose is from 250 μg to 1000 μg. In various aspects, the priming dose is from 250 μg to 500 μg. In various aspects, the priming dose is from 270 μg to 1000 μg.

[0168] In various aspects of the method, two priming doses are administered. In aspects where two priming doses are administered, the first priming dose is from about 150 μg to about 540 μg. In various aspects, the first priming dose is from about 175 μg to about 500 μg. In various aspects, the first priming dose is from about 200 μg to about 400 μg. In various aspects, the first priming dose is from about 200 μg to about 300 μg. In various aspects, the first priming dose is from about 250 μg to about 300 μg. In various aspects, 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 35 μg. In various aspects, the first priming dose is about 270 μg. In various aspects, the first priming dose is 270 μg.

[0169] In aspects where two priming doses are administered, the second priming dose is from about 800 μg to about 1200 μg. In various aspects, the second priming dose is from about 850 μg to about 1150 μg. In various aspects, the second priming dose is from about 900 μg to about 1100 μg. In various aspects, the second priming dose is from about 950 μg to about 1050 μg. In various aspects, 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 various aspects, the second priming dose is about 1000 μg. In various aspects, the second priming dose is 1000 μg.

[0170] In some aspects, two priming doses are administered, the first priming dose is about 270 μg, and the second priming dose is about 1000 μg.

[0171] Table 5 Part 1: Single Therapy Dose Escalation - Group A

[0172]

[0173] Abbreviations: DLT = dose-limiting toxicity; MTD = maximum tolerated dose; N = number of groups; RP2D = recommended phase 2 dose; SMG = safety monitoring group.

[0174] a Dose de-escalation can occur from any dose level (except the initial 30 μg) to improve the determination of the MTD and / or RP2D.

[0175] b A single priming dose will be explored in Groups 4b, 5b, 6a, and 7a. Starting from Group 4b, Groups Na, b, c, etc. can proceed in parallel. Not all dose level groups will be recruited based on emerging safety data.

[0176] C Close recruitment for Group 6c

[0177] d The approximate number of subjects is based on the lack of dose-related toxicity in any group. The actual number of subjects will depend on safety and other findings. In a given subgroup, no more than 3 subjects will be recruited simultaneously.

[0178] e Groups 5d, 6d, and 7d are not part of the dose escalation design but are considered backfills for Groups 5b, 6a, and 7a, respectively. For groups considered backfills, no formal DLT assessment is planned. Once the SMG determines that Group 7a is safe for escalation as guided by BOIN, recruitment can begin into Group 8d.

[0179] When a ≥ Grade 2 adverse event (AE) occurs that is not clearly attributable to the subject or other external causes for the first time, the corresponding group and all subsequent groups in Group A will be expanded to 3 subjects and will follow the standard 3 + 3 dose escalation design. If a DLT occurs in a single subject group, that group will be expanded to 6 subjects. If a second DLT occurs in the group, the immediately preceding dose is the MTD. If only 1 DLT is observed, subsequent groups will proceed according to the 3 + 3 dose escalation design.

[0180] If a response (PR or CR) is observed in a single subject group, subsequent group recruitment will proceed according to the 3 + 3 dose escalation design.

[0181] Table 6 Part 1: Guidelines for Single Therapy Dose Escalation Group A

[0182]

[0183] Abbreviations: DLT = dose-limiting toxicity; MTD = maximum tolerated dose.

[0184] In some aspects, dose escalation will start with a Q2W dosing schedule, which may be changed following cumulative review of safety and PK data and protocol amendment. Administration of the first dose of TNB-486 to the first subject in each cohort must await completion of the first cycle (4 weeks) at the current dose level and review of safety data by the medical monitors (Syneos, Inc. and TeneoTwo) and the principal investigator (or designated sub-investigator; hereinafter referred to as the SMG). Input from biostatistics, PK, and other experts will be sought as necessary.

[0185] In some aspects, based on the SMG's ongoing review of emerging safety and clinical pharmacology data, cohorts receiving intermediate doses between the recommended doses above may be implemented. Additionally, a switch to an alternative dosing schedule may be required. For example, dosing may be switched to a different frequency (e.g., once every 3 weeks), or some cycles may be consistently eliminated from the dosing plan (e.g., the 3rd cycle of each plan may be cancelled). If the dosing schedule is switched to occur more frequently, no dose modification should result in a predicted steady-state concentration (CSS) or Cmax greater than those determined for the next lower dose level. If new intermediate dose levels and / or new dosing schedules are to be evaluated, a protocol amendment with appropriate justification will be submitted.

[0186] Selection of Dose Escalation and MTD / RP2D Doses :

[0187] In some aspects, DLT criteria are used to make decisions regarding dose escalation. Dose escalation decisions will be made by the SMG based on clinically significant toxicity, DLT events, and PK and PD findings (when available). A simple majority of the SMG is required to proceed with dose escalation.

[0188] Dose escalation will proceed as follows (and as shown in Figure 2 ):

[0189] · In a single subject cohort, if the first DLT - evaluable subject in the given cohort completes the safety assessment during the first cycle in the absence of evidence of activity and in the absence of ≥ Grade 2 AEs that are not clearly due to their underlying malignancy or other external causes, escalation to the next recommended dose level will proceed.

[0190] · If a subject in a single subject cohort experiences a non - DLT ≥ Grade 2 AE that is not clearly due to their underlying malignancy or other external causes, the same dose level will be expanded to 3 subjects.

[0191] · If the first 3 DLT - evaluable subjects in a 3 - subject cohort complete the safety assessment during the first cycle without experiencing DLT, progression to the next recommended dose level will proceed.

[0192] If 1 subject at a dose level experiences a DLT (including a single subject cohort), the same dose level will be expanded to 6 subjects.

[0193] If 1 of 6 evaluable subjects experiences a DLT, then the patient will proceed to the next recommended dose level.

[0194] If 2 of 6 subjects experience a DLT, dose escalation will be stopped because the MTD will be exceeded.

[0195] In some aspects, once the SMG safety review of Cycle 1 of Cohort N is completed and if that dose is deemed safe (e.g., the SMG approves further dose escalation or the dose for Cohort N is determined to be the RP2D), any subject remaining on study at the lower dose of TNB-486 may subsequently be treated at the dose assigned to Cohort N (e.g., when the SMG review of Cohort 5 is completed and a decision has been made to escalate to Cohort 6, any subject from Cohorts 1 to 4 who remains on study may have their dose increased to the dose corresponding to Cohort 5). The eligibility of such escalated subjects must be approved by the SMG and will be determined on a case-by-case basis. At a minimum, subjects must have received at least 2 cycles of the current dose of TNB-486 and have undergone at least 1 post-dose disease assessment without any drug-related toxicity that led to a dose reduction to be eligible. Subjects should have stable disease or better and should not have experienced any Grade ≥ 2 AEs that were not clearly due to their underlying disease or other external causes from their most recent dose of TNB-486.

[0196] In some aspects, if available data during the SMG review of Cohort N are unclear for further escalation (e.g., based on the occurrence of non-DLT AEs, plateauing efficacy, suspected RP2D), Cohort N and / or Cohort N-1 may each be expanded to a maximum of 9 subjects at the discretion of the SMG.

[0197] Dose Priming :

[0198] Based on the nonclinical pharmacology of TNB-486, the rate of CRS is expected to be lower than that of other T cell-engaging bispecific antibodies; however, based on the mechanism of action of TNB-486, the most likely DLT remains CRS ≥ Grade 3. Cytokine release syndrome will be managed according to the decision of the principal investigator and institutional guidelines (if institutional guidelines have not yet been established, then in Figure 3 Guidance on administering CRS is provided in .

[0199] In some aspects, if a ≥ grade 3 CRS event occurs in cohort N during the DLT period or at the discretion of the SMG, a dose equal to or less than the dose of cohort N is designated as the 'index dose', and the administration of the dose in subsequent cohorts (cohorts N+1 and onwards) is modified as follows:

[0200] · On cycle 1 day 1, the subject will receive the index dose. The index dose in cohorts N+1 and higher cohorts will not exceed the dose administered in cohort N.

[0201] · In aspects where a second index dose is administered, on cycle 1 day 8, the subject will receive the second index dose.

[0202] · Starting from cycle 1 day 15, the subject will receive the full dose corresponding to the cohort they have been enrolled in.

[0203] · In cohort N+1, the full dose will not exceed the index dose (i.e., the dose administered to cohort N) by more than 50%.

[0204] · The full dose administered in cohorts N+2 and higher cohorts should not exceed +100% of the full dose in the immediately preceding cohort.

[0205] · If 2 ≥ grade 3 CRS AEs occur in the same cohort, the dose escalation of the full dose should not exceed +50% of the immediately preceding cohort.

[0206] · If a DLT occurs, the dose escalation of the full dose should not exceed +33% of the immediately preceding cohort.

[0207] After dosing on cycle 1 day 1, if the subject experiences any ≥ grade 3 toxicity not clearly due to the subject's underlying malignancy or other external causes, the administration of the full dose on cycle 1 day 15 must await approval by the medical monitor. At a minimum, before the full dose is administered, the CRS must have resolved to ≤ grade 1. For such subjects, the dose and timing of the full dose can be modified at the discretion of the medical monitor and the investigator; if the subject has their full dose reduced during cycle 1, the subject will be considered DLT non-evaluable and should be replaced, unless they experience a DLT. In subsequent cycles, the subject will receive the full dose on day 1 and day 15.

[0208] After the implementation of the index dose, if the administration of the full dose is delayed during cycle 1, the DLT period for that subject should be extended by the same amount (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 DLT criteria will be considered DLTs, regardless of whether they occur after the administration of the index dose or the full dose.

[0209] Single Therapy Dose Expansion (Part 2, Group B) :

[0210] In some aspects, the method involves evaluating the MTD (or RP2D) of TNB-486 monotherapy in approximately 30 subjects with biopsy-confirmed RR DLBCL or HGBL. To be eligible for the 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 have a prior treatment history that does not include CD19-targeted therapy. Once the MTD (or RP2D) has been selected based on data from the monotherapy dose escalation (Part 1, Cohort A), Cohort B will be initiated. The SMG will select the MTD (or RP2D) and dosing frequency for Cohort B based on safety, tolerability, and PK / PD data collected during the dose escalation portion of the study.

[0211] Single Therapy Dose Expansion (Part 2, Group C) :

[0212] In some aspects, the method includes evaluating the MTD (or RP2D) of TNB-486 monotherapy in approximately 20 subjects with biopsy-confirmed RR FL (grade 1 to 3a). To be eligible for the 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 have a prior treatment history that does not include CD19-targeted therapy. Once the MTD (or RP2D) has been selected based on data from the monotherapy dose escalation (Part 1, Cohort A), Cohort C will be initiated. The SMG will select the MTD (or RP2D) and dosing frequency for Cohort C based on safety, tolerability, and PK / PD data collected during the dose escalation portion of the study.

[0213] TNB-486 Dose Escalation in Combination with R-CHOP (Part 3, Group D) :

[0214] In some aspects, the method includes evaluating the safety, tolerability, PK, and pharmacodynamic characteristics of TNB-486 administered in combination with R-CHOP every three weeks in approximately 9 to 18 subjects. A schematic study design for Part 3, Cohort D is presented in Figure 1B below.

[0215] All subjects will receive six cycles of TNB-486 plus R-CHOP chemotherapy (cycles 1 to 6) at 21-day intervals, followed by an additional three cycles of TNB-486 and rituximab (cycles 7 to 9), for a total of 9 treatment cycles. R-CHOP will be administered at a fixed dose according to the standard of care. In cycle 1, all subjects will receive R-CHOP on day 1, followed by two escalating 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 start at dose level (DL) 1 (i.e., 270 μg of TNB-486 on day 8 of cycle 1, followed by 1000 μg on day 15 of cycle 1; 7200 μg of R-CHOP plus TNB-486 on day 1 of cycle 2). Based on the DLT distribution, subsequent dose levels will be stepped down or up to DL-1 or DL-2, respectively, according to Table 7.

[0216] Table 7 TNB-486 Dose Levels Tested in Combination with R-CHOP

[0217]

[0218] Abbreviations: R-CHOP = rituximab, cyclophosphamide, doxorubicin, vincristine (Oncovin), prednisone.

[0219] Since common treatment-related toxicities of the T cell engager (i.e., CRS and NT) typically occur within 24 to 72 hours after infusion, in some aspects, the first subject in each cohort will complete at least 1 week of observation after receiving the target dose of TNB-486 plus R-CHOP on day 1 of cycle 2 before additional subjects in that given cohort can receive the target dose. In some aspects, the DLT window will be counted from day 8 of cycle 1 to day 1 of cycle 3 (a total of 3 TNB-486 doses, 35 days).

[0220] In some aspects, subjects in part 3, group D will be recruited according to the 3+3 (i3+3) design, where the target toxicity rate is 30% and the equivalent interval (EI) is (25% to 35%). In all aspects, up to 3 subjects can be recruited simultaneously. In all aspects, the de-escalation or escalation decision will be made based on at least 3 DLT-evaluable subjects per cohort. In all aspects, approximately 9 to 18 subjects will be recruited, assuming 3 dose levels will be studied and 3 to 6 subjects will be assigned at each dose level. In all aspects, the total number of subjects will depend on the number of dose escalations / de-escalations required.

[0221] In all aspects, once the active dose cohort of the combination of TNB-486 and R-CHOP is reached (i.e., the cohort in which at least one partial response or better response is observed), and if the DLT rate of the highest dose cohort within the EI at the time of the SMG meeting (i.e., without mandatory dose reduction), the active dose cohort <N can be expanded to up to 15 subjects at the discretion of the SMG to better evaluate the optimal biological dose / RP2D. In all aspects, if one or more dose levels are expanded, up to 15 subjects will be allocated within the cohort respectively.

[0222] In all aspects, if a DLT attributable to TNB-486 occurs according to the investigator's assessment, drug administration will be stopped. If, in the view of the treating investigator, the patient may derive clinical benefit, TNB-486 treatment can be resumed at a lower dose only after the toxicity has resolved to ≤ Grade 1. In all aspects, after recovery from toxicity, there will be no dose re-escalation of TNB-486, and dose escalation of participants within the cohort will not be permitted. In all aspects, dose adjustment of the R-CHOP component will follow the regular dose modification schedule.

[0223] Selection of the study population:

[0224] In some aspects, patients undergo a screening procedure within 28 days before the initial study drug administration. Adult patients who meet the inclusion criteria and do not meet any of the exclusion criteria will be eligible to participate in the study.

[0225] Inclusion Criteria :

[0226] 1. The subject must be ≥ 18 years old.

[0227] 2. The subject has biopsy-confirmed B-NHL according to World Health Organization (WHO) criteria (Swerdlow 2017).

[0228] 3. Only for Group B: The subject has biopsy-confirmed RR DLBCL or HGBL according to WHO criteria (Swerdlow 2017). At least 15 subjects in Group B should not be treated with CD19-targeted therapy currently.

[0229] 4. Only for Group C: At least 10 subjects in Group C should not be treated with CD19-targeted therapy currently.

[0230] 5. Only for Group D: The subject has biopsy-confirmed, previously untreated CD20-positive DLBCL according to WHO criteria (Swerdlow 2017), including one of the following diagnoses:

[0231] a. DLBCL, not otherwise specified (NOS)

[0232] b. T cell / tissue-rich large B-cell lymphoma

[0233] c. Epstein-Barr virus-positive DLBCL, NOS

[0234] d. ALK-positive large B-cell lymphoma

[0235] e. High-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements (double-hit or triple-hit lymphoma)

[0236] f. High-grade B-cell lymphoma, NOS

[0237] g. DLBCL transformed from indolent lymphoma

[0238] h. Primary cutaneous DLBCL, leg type 1

[0239] i. Follicular lymphoma, grade 3B.

[0240] 6. The subject's B-NHL disease is CD19-positive.

[0241] 7. The subject has ≥1 measurable disease site, as defined in the RECIL 2017 classification. (Younes 2017).

[0242] 8. The subject has received at least 2 lines of therapy, to which the subject is refractory or has relapsed subsequently. To be eligible for the study, the subject must not be a candidate for treatment regimens known to provide clinical benefit in B-NHL.

[0243] 9. Only for Group D: The subject has not received prior treatment for the studied indication (except steroids for lymphoma symptom control) and is eligible for R-CHOP chemoimmunotherapy in the opinion of the investigator.

[0244] 10. Only for Group D: The subject has an IPI score of 2 to 5.

[0245] 11. Only for Group D: The subject has a left ventricular ejection fraction (LVEF) within institutional normal limits, as determined by cardiac echocardiogram (ECHO).

[0246] 12. The subject has an Eastern Cooperative Oncology Group (ECOG) performance status of ≤2.

[0247] 13. The subject must have adequate bone marrow function, defined as: absolute neutrophil count (ANC) ≥1000 / mm 3 ; platelets ≥50,000 / mm 3 (For Part 3 Group D, ≥100,000 / mm 3);Hemoglobin ≥ 8.0 g / dL. Transfusion and / or growth factor support is allowed prior to assessment, but neutrophils, platelets, and hemoglobin must be stable for at least 72 hours after transfusion and / or growth factor administration before screening eligible subjects. If the subject has received 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.

[0248] 14. The subject must have an estimated glomerular filtration rate (eGFR) ≥ 50 mL / min, as estimated by the MDRD formula. Note: According to FDA guidelines, for protein therapeutics > 69 kDa, specifically including antibodies such as TNB-486 (FDA 2010), 'renal impairment is unlikely to alter PK sufficiently to justify dose adjustment'.

[0249] 15. The subject must have total bilirubin < 1.5 × upper limit of normal (ULN; unless the subject has a known diagnosis of Gilbert's syndrome, in which case bilirubin must < 3 × ULN). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) must ≤ 3 × ULN (unless the subject has a known diagnosis of Gilbert's syndrome, in which case AST / ALT must < 5 × ULN).

[0250] Exclusion Criteria :

[0251] If a subject meets any of the following criteria, he / she will not be eligible for treatment: 1. The subject has been diagnosed with or is being treated for another malignancy, the natural history or treatment of which may

[0252] interfere with the safety or efficacy assessment of the study protocol. Patients with existing or concurrent malignancies (the natural history or treatment of which does not have the potential to interfere with the safety or efficacy assessment of the study protocol) are eligible for the trial.

[0253] 2. The subject has a history of CNS involvement of their B-NHL. If CNS involvement is suspected, MRI and / or spinal tap should be performed at screening.

[0254] 3. The subject has a history of leukemic presentation of their B-NHL, except for MCL or MZL.

[0255] 4. The subject has a history or presence of clinically significant CNS pathology (e.g., epilepsy, seizures, stroke, paresis, aphasia, severe brain injury, dementia, neurodegenerative disorders including Parkinson's disease, dementia, cerebellar disease, organic psychiatric syndrome, psychosis or other serious psychiatric illness).

[0256] 5. Subjects have active or history of CNS involvement from autoimmune disease.

[0257] 6. Subjects experienced ≥ grade 3 CRS (based on ASTCT criteria) after prior TCE or CAR T-cell therapy.

[0258] 7. Subjects experienced ≥ Grade 2 neurotoxicity / ICANS (based on ASTCT criteria) following prior TCE or CAR T-cell therapy.

[0259] 8. The subject is at 5×T 1 / 2 or receive another study drug within 28 days of enrollment, whichever is shorter.

[0260] 9. The subject has received peripheral autologous SCT within 12 weeks, or allogeneic SCT within 1 year of the first dose of study drug, or has received SCT and requires continued immunosuppressive therapy.

[0261] 10. Subjects requiring long-term immunosuppressive therapy (including steroids >10 mg prednisone / day); for Part 3, Group D, upfront use of steroids is permitted. Subjects may be eligible if immunosuppressive therapy has been discontinued 14 days or 5 half-lives (whichever is shorter) before the first dose of study treatment, at the discretion of the Medical Monitor and Sponsor.

[0262] 11. In the opinion of the Investigator or Medical Monitor, the subject has any medical or psychiatric condition that places the subject at an unacceptably high risk of toxicity, may interfere with successful or safe treatment delivery, or may interfere with the evaluation of the study drug or the safety of the subject or the interpretation of the study results. Examples include a history of significant mucosal / internal bleeding, severe mental illness, drug abuse (including active alcoholism), active graft-versus-host disease, or known allergic or hypersensitivity reactions to components of the study drug formulation.

[0263] 12. Subjects received any cancer therapy (including radiation, chemotherapy, biologics, cell therapy) or underwent major surgery within 14 days (or within 5 half-lives of anticancer drugs) prior to the first dose of study treatment, whichever is shorter. Chemoreductive chemotherapy is allowed outside of this washout period to reduce the risk of TLS or CRS / NT in subjects with bulky disease.

[0264] 13. The subject has an active infection known to require parenteral antibiotic treatment. After completion of parenteral antibiotics and resolution of symptoms, the subject is considered eligible for the study from the perspective of the infection.

[0265] 14. Subjects with human immunodeficiency virus (HIV) infection, or subjects with chronic or active infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). HIV-infected patients receiving effective antiretroviral therapy and with an undetectable viral load for 6 months are eligible for the trial. Subjects with chronic HBV may be enrolled if the HBV viral load is undetectable on suppressive therapy, or if the subject has a documented cure (negative hepatitis B surface antigen [HBsAg]). Subjects with HCV with a documented cure (HCV RNA undetectable 24 weeks after the end of treatment) may be enrolled.

[0266] 15. Major cardiac abnormalities, such as but not limited to the following: uncontrolled angina or life-threatening unstable arrhythmias, history of myocardial infarction ≤12 weeks before screening, ≥Grade 3 New York Heart Association congestive heart failure, severe cardiac insufficiency, or persistent QTc prolongation (>480 milliseconds, QTc Fridericia).

[0267] 16. If female, the subject must not be pregnant or breastfeeding and must be postmenopausal (for at least 12 consecutive months), or permanently surgically sterilized, or for women of childbearing potential, an effective female contraceptive method as specified in the implementation plan (Section 0) from Day 1 of Cycle 1 through at least 6 months after the last dose of study drug.

[0268] 17. The subject has ≥Grade 2 unremitted adverse events (National Cancer Institute [NCI] Common Terminology Criteria for Adverse Events [CTCAE] v5.0) from prior anticancer treatment, except for the following:

[0269] - Alopecia.

[0270] - Peripheral neuropathy (peripheral neuropathy ≥Grade 3 will be excluded).

[0271] - Anemia or thrombocytopenia (thrombocytopenia must be Grade 4 to trigger exclusion,

[0272] Grade 3 with symptoms or bleeding, or despite transfusion support, not recovered within 72 hours).

[0273] - After consultation with the medical monitor, subjects with irreversible toxicity that is not reasonably expected to be exacerbated by the investigational drug (e.g., hearing loss) may be included. 18. Only for Group D: Current diagnosis of any of the following:

[0274] a. B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma (grey zone lymphoma)

[0275] b. Primary mediastinal (thymic) large B-cell lymphoma

[0276] c. Burkitt lymphoma

[0277] d. HHV8-positive DLBCL, NOS

[0278] e. Primary effusion DLBCL

[0279] 19. For Group D: Contraindications to any of the individual components of R-CHOP, including anthracyclines already received.

[0280] Contraception advice and pregnancy testing: If female, the subject must be postmenopausal, defined as:

[0281] · Age > 55 years, amenorrheic for 12 consecutive months or longer, without alternative medical causes.

[0282] Or

[0283] · Age < 55 years, amenorrheic for 12 consecutive months or longer, without alternative medical causes, and follicle-stimulating hormone level > 40 IU / L.

[0284] Or

[0285] · Permanent surgical sterilization (bilateral oophorectomy, bilateral salpingectomy, or hysterectomy).

[0286] Or

[0287] · For women of childbearing potential, implement an effective contraceptive method from the following list from Cycle 1 Day 1 (or earlier) until at least 6 months after the last dose of investigational drug:

[0288] - Combined (estrogen and progestin) hormonal contraception associated with ovulation suppression

[0289] (oral, intravaginal, transdermal), initiated at least 1 month before Cycle 1 Day 1.

[0290] - Progestin-only hormonal contraception associated with ovulation suppression (oral, injectable, implantable), initiated at least 1 month before Cycle 1 Day 1.

[0291] - Bilateral tubal occlusion / ligation.

[0292] - A partner who has had a vasectomy, provided that the vasectomized partner has had a successful medical evaluation of the surgery and is the sole partner of a female trial participant with reproductive potential.

[0293] - Condom

[0294] - Intrauterine device.

[0295] - Intrauterine hormone-releasing system.

[0296] - True abstinence: when this is in line with the subject's preferred and usual lifestyle,

[0297] avoid heterosexual intercourse (cyclic abstinence [e.g., calendar, ovulation, temperature, post-ovulation methods] and withdrawal are unacceptable).

[0298] If male, from the first dose of the study drug until at least 6 months after the last dose of study treatment, subjects with reproductive potential (i.e., male subjects who have not undergone bilateral vasectomy) must agree to use at least 1 highly effective male contraceptive method (vasectomy or condom) during sexual contact with a female with reproductive potential.

[0299] Treatment Administration :

[0300] In some aspects, TNB-486 is initially administered as an IV infusion Q2W, where 1 treatment cycle will be 28 days. Each dose level in Group A will enroll a minimum of 1 and a maximum of 9 subjects. In the single-agent dose escalation (Part 1, Group A), a starting dose of 30 μg of TNB-486 will be administered as an IV infusion (Q2W), and will be escalated stepwise to a projected maximum of 30,000 μg in subsequent cohorts (Table 5). In Part 2, Groups 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.

[0301] In some aspects, subjects will be premedicated with dexamethasone (10 mg IV) or equivalent. If a subject does not experience an infusion-related reaction (IRR) or immune-mediated toxicity (IMT; e.g., CRS or NT) during a given cycle, the dexamethasone premedication dose can be reduced to 5 mg IV. If a subject does not experience an IRR or IMT during a cycle in which 5 mg of dexamethasone IV is used as premedication, dexamethasone can subsequently be omitted from the premedication regimen. If a subject experiences an IRR or IMT at any time, or if a subject experiences an in-subject dose escalation, they should be premedicated with 10 mg of dexamethasone with the next dose of TNB-486 and tapered as described above.

[0302] In some aspects, 15 minutes to 60 minutes prior to TNB-486 infusion, subjects are routinely premedicated with diphenhydramine (25 mg to 50 mg IV) or equivalent (e.g., cetirizine 10 mg orally [PO] × 1), acetaminophen 650 mg to 1000 mg PO, and ranitidine 150 mg PO / IV or equivalent 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 discretion of the investigator and after approval by the medical monitor.

[0303] In some aspects, the first TNB-486 infusion is given over 2 hours (±10 minutes). After selection of the RP2D, the duration of the infusion can be shortened. After the infusion on Day 1 of Cycle 1, subjects will be admitted as inpatients for 48 hours; if a subject receives a priming dose at C1D1 or experiences ≥ Grade 2 CRS with their first dose, they will also be admitted for 48 hours at C1D15. In subsequent cycles, subjects are closely monitored for 2 hours after each infusion. For more details on dose modification, see the pharmacy manual.

[0304] In some aspects, the doses selected for evaluation in the single-agent dose expansion (Part 2, Groups B and / or C) can be doses that are equal to or lower than the MTD or RP2D defined in the single-agent dose escalation (Part 1, Group A). Based on safety, tolerability, and PK / PD data collected from the single-agent dose escalation (Part 1, Group A), in consultation with the SMG, the Sponsor will select the dosing frequency for the single-agent dose expansion (Part 2, Groups B and / or C).

[0305] Preparation / Reconstitution of Dosage Forms :

[0306] In some aspects, TNB-486 drug product (activity) is provided as a solution in a bottle, prepared at 2 mg / mL, with each bottle having an 8 mL extractable volume of drug product, and is administered by IV infusion. For the first 4 groups (Part 1, Group A; Table 5) in a monotherapy dose escalation, TNB-486 will be diluted in 2 steps. The first dilution step uses the non-DEHP 50 mL IV bag provided by the test kit to reduce the intensity of TNB-486 by 100 times to 20 μg / mL. The second dilution step requires that the pre-diluted TNB-486 of the specified volume be transferred dose-dependently to a non-DEHP 100 mL IV bag (100 mL containing non-DEHP IV bags will be provided by TeneoTwo). The final concentrations of the first 4 dosage groups are 240 ng / mL, 720 ng / mL, 2.16 μg / mL, and 6.48 μg / mL, respectively. The diluent for each dilution step is saline with IV stabilizer solution (IVSS) 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 a 15 mL extractable volume. IVSS vials are formulated at 10× strength and have a 1× working strength in the IV bag.

[0307] In some aspects, the study drug for dose cohorts 5 and higher (Table 5) will be prepared in a single dilution step, where a dose-dependent volume of TNB-486 is transferred directly from the drug product vial into a 100 mL non-DEHP IV bag provided with each kit. The diluent is saline with IVSS added prior to the addition of the active TNB-486 drug product, with the formulation being identical compared to the lower dose cohorts. In dose cohorts 5 and higher, the concentration of active drug product ranges from 24.3 μg / mL to 303.7 μg / mL.

[0308] 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°C to 25°C) should not exceed 6 hours (or 24 hours at 2°C to 8°C) to minimize the risk of degradation and microbial contamination of the drug product. Storage time may be updated as additional sterility / stability data become available.

[0309] In some aspects, the total volume administered at each dose is 250 mL.The infusion rate is controlled using an infusion pump and their respective DEHP-free infusion sets containing an inline filter.

[0310] In some aspects, the TNB-486 drug product vials are stored at 5 ± 3°C. The IVSS vials are stored at ambient temperature. The diluted active pharmaceutical ingredient is tested at the lowest (240 ng / mL) and highest doses (303.7 μg / mL), where the diluted active pharmaceutical ingredient is exposed to light for up to 6 hours at controlled room temperature (20°C to 25°C) and for up to 24 hours at 2°C to 8°C for infusion device compatibility.

[0311] Eastern Cooperative Oncology Group (ECOG) performance status:

[0312] Aspects of the present disclosure include evaluating the ECOG performance status of subjects at various time points throughout the study. The ECOG performance status is recorded using the scoring method in Table 8.

[0313] Table 8: Eastern Cooperative Oncology Group (ECOG) Performance Status

[0314]

[0315] Pregnancy Testing :

[0316] Aspects of the present disclosure include obtaining serum or urine for pregnancy testing of women of childbearing potential when screened by the central laboratory, and the serum or urine pregnancy test must be performed locally within 72 hours of dosing with a negative result. Thereafter, a urine pregnancy test must be performed locally (confirmed by serum pregnancy test if positive) and a negative result recorded before dosing at the start of each cycle.

[0317] Clinical Laboratory Tests :

[0318] Aspects of the present disclosure include obtaining samples for the clinical laboratory tests outlined in Table 9 at least at screening and locally at subsequent visits, the EOT visit, and the 90-day follow-up visit, as outlined in the schedule of events (all groups).

[0319] In some aspects, a certified laboratory is utilized to process and provide the results of the clinical laboratory tests. Laboratory reference ranges are obtained prior to the start of the study. The baseline laboratory test results for the clinical evaluation of a specific test are defined as the last measurement before the initial dose of TNB-486.

[0320] Table 9: Clinical Laboratory Tests

[0321]

[0322]

[0323] Abbreviations: Ab = antibody; aPTT = activated partial thromboplastin time; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; GGT = γ-glutamyl transferase; 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 cell; TNF = tumor necrosis factor; WBC = white blood cell;

[0324] Biomarker Assessment :

[0325] Imaging Assessment :

[0326] In aspects of the present disclosure, for all subjects, baseline disease assessment using PET-CT or CT in the case of non-FDG avid diseases must be performed within 28 days before the first dose of the study drug. In aspects, imaging will be performed on the first day of every 3rd cycle and repeated as clinically indicated. Disease assessment may be performed by a central imaging provider. See Table 10 for examples of details and timing of disease response assessment.

[0327] Tumor Tissue :

[0328] In aspects of the present disclosure, at screening, all subjects should provide 14 to 15 unstained tissue sections (e.g., from core, excision, or fine needle aspiration; if possible, at least 10 on charged slides and 4 to 5 on uncharged slides) or formalin-fixed paraffin-embedded blocks containing sufficient tumor to cut 14 to 15 sections from its most recent biopsy (if available). The collection date and a complete pathology report (including results of flow cytometry, cytogenetics / FISH, and molecular testing, if performed) should be submitted with the sample. If the subject has previously received anti-CD19 therapy, biopsy material collected after disease relapse or recurrence following CD19-targeted therapy should be sent. In aspects, tumor samples will be analyzed at the molecular and cellular levels to determine how baseline biomarker levels and changes relative to baseline are associated with clinical outcomes, safety, and resistance.

[0329] In some aspects, for Part 2, subjects in Groups B and C are required to provide optional fresh tumor biopsies at screening, on Day 1 of Cycle 3, and at progression to evaluate exploratory biomarkers (e.g., next-generation sequencing to identify druggable mutations). In aspects, the goal will be to enroll 10 or more subjects who agree to these exploratory biomarker analyses in addition to mandatory biopsies / aspirations under suspected CR and, if possible, under suspected progression.

[0330] In some aspects, subjects in Part 2, Groups B and C may be required to provide optional fresh tumor biopsies at screening, Day 1 of Cycle 3, and at progression to evaluate exploratory biomarkers (e.g., next-generation sequencing to identify druggable mutations). In all aspects, the goal will be to enroll 10 or more subjects who will consent to these exploratory biomarker analyses in addition to mandatory biopsies / aspirations under suspected CR and, when possible, under suspected progression.

[0331] Table 10 Disease Response Assessment

[0332]

[0333] Abbreviations: CR = complete response; EOT = end of treatment; PD = disease progression; PET-CT = positron emission tomography-computed tomography.

[0334] a Subjects in Groups B and C will be required to provide optional fresh tumor biopsies at screening, C3D1, and suspected progression.

[0335] b Subjects who discontinue treatment for reasons other than disease progression or withdrawal of consent should continue to have their disease response evaluated every 12 weeks (±3 weeks) for the first 12 months, then every 26 weeks (±4 weeks) until documented disease progression, the start of subsequent anticancer agent therapy, or withdrawal.

[0336] c The EOT visit should occur within 30 days after the last dose of TNB-486. If alternative therapy is initiated during this period,

[0337] then the EOT visit should be conducted prior to the first dose of alternative therapy. After the last dose of the study drug, adverse events and concomitant medications / treatments should be followed for 90 days, or until the subject starts a new line of therapy, whichever occurs earlier.

[0338] Subjects' survival will be tracked by phone call every 12 weeks starting from the last visit. Once reliable half-life data for TNB-486 are available and if T 1 / 2 exceeds 18 days, the 90-day post-treatment final visit can be conducted at a time further from the last dose so as to capture PK and ADA data at T ≥ 5 × T 1 / 2 ; if this occurs, AEs will be collected until the time of that final visit or until the start of a new line of therapy, whichever occurs earlier.

[0339] dIf bone marrow involvement of a subject's disease is suspected at screening in the absence of a prior medical history, a bone marrow biopsy should be performed. If CR is suspected in subjects with a history of bone marrow involvement and / or when a biopsy is clinically indicated while the subject is in the study, bone marrow samples should be provided to the central laboratory for disease response assessment.

[0340] e Disease assessments will be centralized.

[0341] Blood Samples :

[0342] In aspects of the present disclosure, blood samples will be collected from all subjects at the time points indicated in the event schedule (all groups) to assess PK, PD, and response biomarkers, as well as anti-drug antibodies (ADA).

[0343] In some aspects, blood samples will also be collected at specified time points throughout the study to assess biomarkers (i.e., cytokines and exploratory biomarkers [such as circulating free DNA]; Table 11). Additionally, any remaining tissue or body fluid samples obtained from the subject during the study or standard of care procedures, with the subject's permission, may be used to assess exploratory biomarkers.

[0344] Table 11 Schedule for Blood Sample Collection (All Groups)

[0345]

[0346] Abbreviations: ADA = anti-drug antibody; EOI = end of infusion; EOT = end of treatment; min = minute; N / A = not applicable; PK = pharmacokinetics; Unsched = unscheduled.

[0347] f If possible, samples collected at the same time point will be included in a single blood draw. Blood samples for PK, biomarker assessment, and ADA testing will be shipped to the central laboratory.

[0348] g Pharmacogenomics is optional and should only be collected at screening. Separate consent is required.

[0349] h PK, ADA, and biomarker (e.g., cytokines, circulating free DNA) tests will be analyzed centrally in batches. Additional ADA testing will be performed during PK sampling time points, as appropriate.

[0350] i Unscheduled visits may occur at any time during the study. The study activities shown will be conducted at the discretion of the investigator.

[0351] j Once sufficient half-life data are available for TNB-486, and if T 1 / 2 exceeds 18 days, the last treatment post-visit at 90 days can be conducted at a time corresponding to approximately 5×T 1 / 2 to capture PK and ADA data when T ≥ 5×T 1 / 2 .

[0352] ADA will be collected only on Day 1 of each cycle, except in Cycle 1, in which ADA samples will also be collected on Day 15.

[0353] Exploratory Study Samples :

[0354] In aspects of the present disclosure, serum samples for biomarker analysis are collected from all subjects.

[0355] Residual Blood and Tissue Samples :

[0356] In aspects of the present disclosure, existing collected residual blood and tissue samples for biomarkers, PK, and tissue samples can be used for optional exploratory studies.

[0357] Pharmacogenomic Samples :

[0358] In aspects of the present disclosure, optional whole blood samples for DNA and RNA isolation are collected at the time of screening or before dosing on Day 1 of Cycle 1.

[0359] Assessment / Variables of Activity, Pharmacokinetics, Pharmacodynamics, Immunogenicity, Pharmacogenetics, and Safety :

[0360] Activity Variables :

[0361] In aspects of the present disclosure, activity will be measured by the change in target lesions according to the RECIL 2017 criteria (see Table 12).

[0362] In some aspects, activity endpoints (determined using the 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 progression (TTP), time to response (TTR), and duration of objective response (DOR).

[0363] Table 12 Activity Measurements

[0364]

[0365] Abbreviations: CR = complete response; CT = computed tomography; FDG-PET = [18F]2-fluoro-2-deoxy-D-glucose; PR = partial response;

[0366] Pharmacokinetic Variables :

[0367] In aspects of the present disclosure, values of the PK parameters of TNB-486, including C max , to C max (T max ), area under the concentration-time curve (AUC t ) from time 0 to the time of the last measurable concentration, CL, terminal elimination rate constant, and t 1 / 2 ), will be determined using non-compartmental methods after injection in Cycle 1. Additional analyses will be performed if deemed useful and appropriate.

[0368] Drug Concentration Measurements :

[0369] In aspects of the present disclosure, the time of collection of each blood sample will be recorded to the nearest minute in the source document and the appropriate eCRF. In aspects, the date and start / end time of the TNB-486 infusion for the subject should also be recorded in the appropriate eCRF to the nearest minute at the time of PK sampling.

[0370] Based on the PK data from the single-agent dose escalation (Part 1, Group A), the pharmacokinetic time points may be slightly altered for the single-agent dose expansion (Part 2, Groups B and C).

[0371] Collection of Blood Samples for TNB-486 Pharmacokinetic Determination :

[0372] In aspects of the present disclosure, for subjects in all groups of the study, a single blood draw at the designated time points will permit TNB-486 PK analysis (see Table 13 and below). In aspects, samples should not be drawn from the same group that received TNB-486. In aspects, for the Q2W dosing regimen, samples will be collected by venipuncture into appropriately labeled evacuated serum collection tubes at the following time points. If a significant AE is observed, then additional unscheduled PK samples may be drawn.

[0373] Table 13

[0374]

[0375] Collection of Blood Samples for Anti-Drug Antibody (ADA) Determination :

[0376] In aspects of the present disclosure, samples for ADA will be drawn before dosing TNB-486 on Day 1 of each cycle and before dosing on Day 15 of Cycle 1.

[0377] Exploratory Study Variables :

[0378] In some aspects, exploratory studies will be conducted to investigate exposure-response relationships through the relationship of biomarkers with PK, safety, and clinical activity. In all aspects, samples will be collected for exploratory studies of known and novel biomarkers. The types of biomarkers to be analyzed may include, but are not limited to, nucleic acids, proteins, lipids, or metabolites. The samples may be analyzed as part of a post hoc assessment of factors affecting the subject's response to the investigational drug or the development and progression of the subject's disease or related medical conditions. The samples may also be used to develop new diagnostic tests, therapies, research methods, or technologies.

[0379] Safety Variables :

[0380] In all aspects of the present disclosure, adverse events, laboratory profiles, physical examinations, and vital signs will be evaluated throughout the study. In all aspects, adverse events will be graded according to NCI-CTCAE version 5.0.

[0381] Appropriateness of Measurements :

[0382] In all aspects of the present disclosure, standard PK, statistical, clinical, and laboratory procedures will be utilized in this study. In all aspects, blood will also be drawn for PD markers, which can add useful information for selecting the appropriate dose of TNB-486 for future studies. Archived tissues may also be used to select the appropriate dose of TNB-486 for future studies and to select the appropriate subject population for treatment.

[0383] Determination of the Maximum Tolerated Dose :

[0384] In all aspects of the present disclosure, if the MTD is identified, it will be defined as the highest dose level at which < 2 out of 6 subjects experience DLT.

[0385] Determination of the Recommended Phase 2 Dose :

[0386] In all aspects of the present invention, if the MTD is reached, the RP2D will not be a dose higher than the MTD and will be selected based on the totality of the data by pooling and evaluating all available data on target engagement, clinical PK, PD, activity, and safety of TNB-486.

[0387] Toxicity Management :

[0388] In aspects of the present disclosure, for the purposes of medical management, all AEs and laboratory abnormalities occurring during the study period must be evaluated by the investigator. The clinical toxicity grading scale from NCI-CTCAE version 5.0 (available on the CTEP homepage at http: / / ctep.info.nih.gov) will be used for the grading of AEs and laboratory abnormalities reported as AEs, each of which will follow a satisfactory clinical resolution. TNB-486 has not been clinically tested in humans, and thus the AE profile in humans is unknown.

[0389] In aspects, AEs should be classified as pre-existing or treatment-emergent (TEAEs):

[0390] · A TEAE is defined as an AE that was not present prior to the start of TNB-486 treatment, or an AE that was present prior to the start of TNB-486 treatment and worsened in intensity and / or frequency after the start of TNB-486 treatment.

[0391] · A pre-existing AE is an AE that does not meet these criteria. Subjects with pre-existing AEs should be evaluated for any underlying disease or other cause and treated appropriately.

[0392] In aspects, co-morbidities or other causes of subjects with TEAEs should also be evaluated and treated appropriately. If a TEAE is clearly due to the subject's underlying malignancy or other external cause, after discussion with the medical monitor, the administration of TNB-486 can be modified at the discretion of the principal investigator. If a TEAE is not clearly due to the subject's underlying malignancy or other external cause, the dose of TNB-486 can be modified as follows, regardless of whether there is a'reasonable likelihood' that the TEAE is TNB-486-related:

[0393] · For subjects who have completed Cycle 1 and have at least clinically or radiologically stable disease (SD, MR, PR, or CR) but have experienced a reversible TEAE, the dose of the study drug can be delayed up to 28 days after the scheduled dosing date. Subjects with a dosing delay > 28 days should be discontinued from the study.

[0394] · During any cycle, if a subject develops ANC < 500 / μL, platelets

[0395] < 10,000 / μL or hemoglobin < 6.5 g / dL, blood samples must be collected every 3 days and study treatment must be stopped. When ANC recovers to ≥ 1,000 / μL and platelet count recovers to

[0396] ≥50,000 / μL or the baseline platelet count level (in subjects with baseline platelets <50,000 / μL), or when hemoglobin has recovered to ≥8.0 g / dL, treatment may resume.

[0397] · In any subject who experiences CRS, TLS, or a non-hematologic TEAE that meets the equivalent criteria for DLT, TNB-486 should be interrupted until the toxicity has resolved to ≤Grade 1 or baseline; thereafter, with the approval of the medical monitor, the study drug may be resumed at a reduced dose level.

[0398] Up to 2 dose reductions are permitted to manage toxicity in all aspects; thereafter, the toxicity will be considered 'unacceptable' and the subject will be withdrawn from treatment.

[0399] Although the investigator's decision should be used for subject management regarding toxicity, guidelines for managing CRS and NT are provided herein.

[0400] Definition of Dose-Limiting Toxicity (DLT) for Dose Escalation :

[0401] In all aspects of the present disclosure, the DLT observation period for dose escalation purposes is 28 days and encompasses the first complete treatment cycle of TNB-486 (2 doses of TNB-486). Regular conference calls will be held between the SMGs to review / confirm potential DLTs, assess AEs, and evaluate laboratory abnormalities. In all aspects, events occurring outside the DLT window may be evaluated during these calls when making dose escalation decisions. In all aspects, DLT is defined as a TEAE that is not clearly due to the subject's underlying malignancy or other external causes and meets the following criteria.

[0402] In aspects of the present invention, DLT evaluable subjects are defined as the following subjects :

[0403] · Received at least 1 dose of TNB-486 and experienced DLT within the first 28 days

[0404] or

[0405] · Received at least 2 doses of TNB-486 on a Q2W schedule and had toxicity evaluated during the 28-day evaluation period

[0406] In all aspects, NCI-CTCAE version 5.0 will be used. The DLT definition is provided below.

[0407] Non-hematological Dose-Limiting Toxicity :

[0408] · Non-hematologic AEs ≥Grade 3, with the following exceptions:

[0409] - Grade 3 or 4 isolated electrolyte abnormalities (i.e., those without clinical consequences) that resolve to ≤Grade 1 within 72 hours (with or without intervention).

[0410] - Grade 3 hypoglycemia / hyperglycemia responsive to optimal medical management within 72 hours.

[0411] - Grade 3 nausea / vomiting / diarrhea responsive to optimal medical management within 72 hours.

[0412] - Alopecia or vitiligo of any grade.

[0413] - Grade 3 fatigue lasting < 10 days.

[0414] · Any AE that requires a delay of > 28 days in the initiation of the next planned cycle.

[0415] · Hepatotoxicity meeting the Hy criteria.

[0416] Hematological Dose-Limiting Toxicity :

[0417] · Grade 3 CRS or Grade 4 CRS that does not resolve to ≤ Grade 1 within 72 hours.

[0418] · Grade 3 TLS or Grade 4 TLS that does not resolve to ≤ Grade 1 within 72 hours.

[0419] · Grade 4 neutropenia lasting > 5 days or febrile neutropenia.

[0420] · Grade 3 thrombocytopenia with bleeding or Grade 4 thrombocytopenia.

[0421] · Grade 4 anemia.

[0422] · Grade 5 AE.

[0423] · Lymphopenia is not considered a DLT.

[0424] · Adverse events that require a delay of > 28 days in the initiation of the next planned cycle.

[0425] Cytokine Release Syndrome (CRS) Dose-Limiting Toxicity :

[0426] Cytokine release syndrome, with or without neurotoxicity, is the major toxicity associated with T cell redirection therapies (CAR and T-BsAbs / BiTE). CRS occurs due to overactivation of the immune system and is mediated primarily by the secretion of pro-inflammatory cytokines (most importantly IL-6 and IL-1). Signs and symptoms are those of systemic inflammation and include the following: high fever / chills, hypotension, hypoxia, neurological changes, pain, nausea, and headache. Cytokine release syndrome can present with varying degrees of severity, from fever and flu-like symptoms to refractory hypotension requiring high-dose vasopressors and organ damage. Meta-analyses have shown that clinical findings, particularly fever, are usually the first indicator of CRS onset (Hay 2017; Wang and Han 2018).

[0427] CRS historically has occurred within 14 days of the first CAR / T-BsAb administration and generally does not occur in subsequent cycles. If CRS occurs, low or selective activation of TNB-486 can delay CRS.

[0428] 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 Treatment guidelines are provided that can be used for subject management; however, if CRS management guidelines exist, it is recommended that investigators follow the institutional guidelines for CRS management.

[0429] Table 14 According to Lee 2019 Guidelines for Cytokine Release Syndrome Grading

[0430]

[0431] Abbreviations: BiPAP = bilevel positive airway pressure; CPAP = continuous positive airway pressure; CRS = cytokine release syndrome. k Fever is defined as a temperature ≥38°C not attributable to any other cause. In subjects with CRS who then receive antipyretics or anti-cytokine therapies, such as tocilizumab or steroids, fever is no longer required to grade subsequent CRS severity. In such cases, CRS grading is driven by hypotension and / or hypoxia.

[0432] 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 1 vasopressor, and hypoxia requiring low-flow nasal cannula is classified as grade 3 CRS.

[0433] mLow-flow nasal cannula is defined as oxygen delivered at 6 L / minute. Low-flow also includes leaky oxygen delivery sometimes used in pediatrics. High-flow nasal cannula is defined as oxygen delivered at >6 L / minute.

[0434] Source: Lee 2019

[0435] Neurological Dose-Limiting Toxicity :

[0436] The etiology of NT is unclear but has been hypothesized to stem from endothelial activation / microangiopathy, possibly downstream of IL-1 secretion by monocytes / macrophages (Gust 2017; Giavidris 2018; Norelli 2018). Onset usually occurs with or after CRS (most CRS grades ≥3). Isolated NT after administration of anti-CD19 T-BsAb has been described (Velasquez 2017). Early symptoms of NT include tremors, dysgraphia, expressive aphasia, impaired attention, and somnolence; subsequent progression may lead to delirium, headache, restlessness, cerebral edema, ataxia, confusion, seizures, and coma.

[0437] 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 can be used to grade NT (Table 15; Lee 2019).

[0438] Table 15: Consensus Grading of American Society for Transplantation and Cellular Therapy Adult Immune Effector Cell-Related Neurological Toxicity Syndrome According to Lee et al., 2019 Table 16 Brain Disease Assessment Tool for Grading Immune Effector Cell-Related Neurological Toxicity Syndrome According to Lee 2019

[0439]

[0440]

[0441] Abbreviations: EEG = electroencephalogram; ICANS = immune effector cell-associated neurotoxicity syndrome; ICE = immune effector cell-associated encephalopathy; ICP = intracranial pressure; NA = not applicable.

[0442] Note: The ICANS grade is determined by the most severe event not attributable to any other cause (ICE score, level of consciousness, seizures, motor findings, elevated ICP / brain edema); for example, a subject with an ICE score of 3 with generalized seizures is classified as grade 3 ICANS.

[0443] a If a conscious subject with global aphasia has an ICE score of 0, they can be classified as grade 3 ICANS, but if they cannot be awakened, a subject with an ICE score of 0 can be classified as grade 4 ICANS.

[0444] b A low level of consciousness should be attributed to no other cause (e.g., no sedative medications).

[0445] c Tremors and myoclonus associated with immune effector cell therapy can be graded according to CTCAE v5.0, but they do not affect ICANS grading.

[0446] d Intracranial hemorrhage with or without associated edema is not considered a neurotoxicity feature and is excluded from ICANS grading. It can be graded according to CTCAE v5.0.

[0447] Source: Lee et al., 2019

[0448] Neurological AEs in subjects receiving TNB-486 require frequent CAR-T cell therapy-related toxicity (CARTOX-10) or immune effector cell encephalopathy (ICE) examinations (Table 16) and neurological examinations. Early neurological consultation, use of anti-epileptic drugs, and intensive care unit / airway support as needed are encouraged. Table 17 provides treatment guidelines that can be used for subject management. However, if available, investigators are recommended to follow institutional guidelines for CRS management.

[0449] Table 17 Recommended Guidelines for Managing Neurological Toxicity Confounding Dose-Limiting Toxicity

[0450]

[0451]

[0452] Abbreviations: CARTOX-10 = chimeric antigen receptor toxicity; ICE = immune effector cell encephalopathy.

[0453] Note: CARTOX-10 (left column) has been updated to the ICE tool (right column). The ICE addition commands follow the assessment to replace 1 of the CARTOX-10 directed questions. The scoring system remains the same.

[0454] Note: Scoring: 10, no impairment; 7 - 9, grade 1 ICANS; 3 - 6, grade 2 ICANS; 0 - 2, grade 3 ICANS; 0, grade 4 ICANS due to the subject being unable to be awakened and ICE assessment not being possible.

[0455] Source: Lee 2019

[0456] Activity Analysis

[0457]

[0458] Abbreviations: AE = adverse event; CT = computed tomography; EEG = electroencephalogram; ICU = intensive care unit; IV = intravenous; LP = lumbar puncture; MRI = magnetic resonance imaging.

[0459] Objective Response Rate (ORR) :

[0460] At the discretion of the investigator, any toxicity considered related to the investigational drug was considered grounds for stopping the drug. Other AEs could be considered DLTs determined by the TeneoTwo medical monitor in conjunction with the investigator.

[0461] All decisions regarding continuous dosing of individual subjects will be appropriately medically managed by the investigator in conjunction with the medical monitor(s). These decisions will be driven by the DLT criteria described above.

[0462] Any subject who did not complete the full 28-day DLT observation period for any reason other than DLT will be considered non-DLT evaluable for dose escalation and / or MTD assessment and will be replaced at the same dose level. In the absence of DLT, additional subjects may be recruited at a given dose level to explore factors influencing AEs or to accumulate additional safety data.

[0463] Progression-Free Survival (PFS) :

[0464] In aspects of the present disclosure, for the cohorts in single-agent dose escalation (Part 1, Group A), the activity data will be tabulated. For single-agent dose expansion (Part 2, Groups B and C), activity analyses will be performed as needed based on the EE population and the safety population. In aspects, the RECIL 2017 criteria will be used to determine subject response and disease progression.

[0465] In aspects of the present disclosure, the objective response rate, DOR, PFS, and CBR for single-agent dose escalation (Part 1, Group A) and single-agent dose expansion (Part 2, Groups B and C) will be summarized and tabulated. Kaplan-Meier estimates of the relevant CIs for PFS and median PFS, OS, and TTP will be provided. Additionally, in some aspects, a descriptive summary of the ORR and its DOR and CBR and the relevant CIs will be provided. In aspects, for single-agent dose expansion (Part 2, Groups B and C), activity analyses will be performed based on the efficacy-evaluable population and repeated for the safety population, unless the sample sizes of the two populations are the same. Furthermore, in some aspects, the results from single-agent dose escalation (Part 1, Group A) at the MTD or RP2D may be combined with the results from single-agent dose expansion (Part 2, Groups B and C) for appropriate analyses.

[0466] Duration of Objective Response (DOR):

[0467] In aspects of the present disclosure, the objective response rate is defined as the proportion of subjects with a confirmed partial or complete response to treatment, and is sometimes referred to as the overall response rate. In aspects, the ORR for each dose cohort will be estimated using all sites combined. In aspects, the Clopper-Pearson method, as well as the best overall response (CR, PR, SD, PD), will also be used to summarize the two-sided 80% and 90% exact binomial CIs for the ORR.

[0468] Clinical Benefit Rate (CBR) :

[0469] In aspects of the present invention, progression-free survival time is defined as the time from the first dose of TNB-486 to progression or death (whichever occurs first). In aspects, if no event occurs, the subject will be censored at the date of the last tumor assessment. In aspects, the Kaplan-Meier method will be used to analyze PFS.

[0470] Clinical Laboratory Tests :

[0471] In aspects of the present disclosure, the duration of objective response of a subject is defined as the time from the initial objective response to disease progression or death (whichever occurs first). In aspects, if the subject does not progress or die, then the subject will be censored at the date of the last tumor assessment, similar to the censoring rules for PFS analysis.

[0472] In aspects, the DOR will be analyzed in the same manner as the PFS analysis.

[0473] Eastern Cooperative Oncology Group (ECOG) Performance Status :

[0474] In aspects of the present disclosure, the clinical benefit rate is defined as the proportion of subjects with a confirmed CR, PR, or MR or SD at least 24 weeks after responding to treatment. In aspects, the CBR for each group will be estimated using all sites combined. In aspects, the Clopper-Pearson method will also be used to summarize the two-sided 80% exact binomial CI for the CBR.

[0475] Vital Signs and Physical Examinations :

[0476] In aspects of the present disclosure, baseline laboratory tests will be performed at the central laboratory used for the study. In aspects, all laboratory tests for disease response assessment from subsequent time points will be performed at the central laboratory. In aspects, all samples for PK, ADA, and biomarker assessment will also be managed by the central laboratory. In aspects, all other laboratory tests will be performed at local laboratories associated with the clinical sites.

[0477] In aspects of the present disclosure, changes relative to baseline in clinical laboratory results will be summarized by treatment group and time point using descriptive statistics. In aspects, a summary of the changes from baseline to the last available visit will be provided. In aspects, the changes will be calculated as the proportion of subjects with values below, at, or above the normal range at the final visit, relative to the proportion of subjects with values below, at, or above the normal range of a specific laboratory test at baseline.

[0478] In aspects of the present disclosure, laboratory abnormalities meeting NCI-CTCAE version 5.0 and treatment-emergent laboratory abnormalities will be summarized by treatment group and overall.

[0479] Pharmacokinetics :

[0480] In aspects of the present disclosure, ECOG performance scores will be listed in the subject data listings and summarized by visit and by change relative to baseline across visits.

[0481] Tables and Summary Statistics :

[0482] In aspects of the present disclosure, changes relative to baseline in vital signs and physical examination findings will be summarized by treatment group and time point using descriptive statistics.

[0483] Dose Proportionality Analysis :

[0484] Missing Values and Model Violations :

[0485] In aspects of the present invention, serum concentrations and PK parameter values of TNB-486 will be tabulated for each subject, for each dose level, and summary statistics will be calculated for each sampling time and each parameter.

[0486] Pharmacodynamics / Biomarkers :

[0487] In aspects of the present disclosure, the pharmacokinetic parameters of TNB-486 from a specific dosing regimen evaluated on Day 1 of Cycle 1 will be analyzed as follows. Dose-normalized Cmax and dose-normalized AUC will be analyzed, provided that they can be adequately determined from the data. The model used for statistical analysis will include the dose level of TNB-486 as a categorical variable. Covariates such as age, race, sex, and other covariates that may 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 α level of 0.10, the covariate may be removed from the model.

[0488] In various aspects, natural logarithmic transformation will be used for Cmax and AUC unless the data clearly indicate that another transformed or untransformed variable provides a more symmetrical probability distribution and / or a more uniform variance across dose levels. In various aspects, assuming that at least 3 dose levels of TNB 486 are studied, contrasts in dose level effects selected to be sensitive to an approximately linear function of dose or the logarithm of dose will be tested.

[0489] Additional analyses will be performed if useful and appropriate.

[0490] V. Drug Compositions for Use :

[0491] In all aspects of the present disclosure, all available data will be included in the dose proportionality analysis. Data exclusions, if any, will be documented and justification provided.

[0492] In various aspects, the values of PK variables are usually determined by simply using the available data (C max , AUC, etc.) without replacing the missing individual concentration value. However, if the missing individual concentration results in a PK parameter value that is likely too low or too high to be meaningful, the value of the PK parameter will be temporarily considered missing. In this case, the value of the missing individual concentration can be estimated so that the appropriate value of the PK parameter can be included in the analysis. In various aspects, the estimated value will be obtained using an appropriate method that takes into account the individual characteristics of the subject.

[0493] In all cases, if outliers are identified and / or significant non-normal probability distributions are observed (in C max If the data are not available for analysis (after logarithmic transformation of the AUC and AUC), nonparametric analyses may also be performed. Such model violations can be identified by graphical methods, measures of non-normality (e.g., skewness, kurtosis), or other appropriate methods. If different dose levels have unequal variances such that conclusions may be affected, approximations that allow for unequal variances will be used. The possibility of bias due to missing data from subjects who discontinued prematurely due to adverse events will be addressed.

[0494] Example 1 :

[0495] In various aspects of the present disclosure, biomarker analysis is exploratory. In various aspects, descriptive statistics of baseline, post-baseline, and changes relative to baseline biomarkers will be summarized and listed by measurement time point / visit. In addition, exploratory analyses will be performed to assess the association of each biomarker or combination of biomarkers with clinical outcomes, the regulation of biomarkers related to mechanism of action, and biomarkers or combinations of biomarkers that potentially predict therapeutic response.

[0496] ​

[0497] Aspects of the present disclosure include pharmaceutical compositions for treating B-cell non-Hodgkin lymphoma in a patient, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486, wherein TNB-486 is administered according to a 28-day treatment cycle, and wherein the therapeutically effective amount of TNB-486 in the pharmaceutical composition is from about 30 μg to about 30,000 μg.

[0498] In some aspects of the pharmaceutical composition for use, the treatment cycle is repeated two or more times. In some aspects, TNB-486 is administered to the patient as a single therapy. In some aspects, TNB-486 is administered by intravenous infusion (IV). In some aspects, the patient has received at least two prior lines of systemic therapy. In some aspects, the patient is CD19 positive. In some aspects, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of less than or equal to 2. In some aspects, the patient has adequate bone marrow function. In some aspects, the patient has an estimated glomerular filtration rate (eGFR) of greater than or equal to 50 mL / min. In some aspects, the subject has a total bilirubin of less than or equal to 1.5 times the upper limit of normal, an aspartate aminotransferase (AST) of less than or equal to 3 times the upper limit of normal, and an alanine aminotransferase (ALT) of less than or equal to 3 times the upper limit of normal.

[0499] Aspects of the present disclosure include pharmaceutical compositions for improving the objective response rate (ORR) in a patient diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, and wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0500] Aspects of the present disclosure include pharmaceutical compositions for improving the overall survival (OS) rate in a patient diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, and wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0501] Aspects of the present disclosure include pharmaceutical compositions for improving the progression-free survival (PFS) rate in a patient diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered according to a 28-day treatment cycle, and wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0502] Aspects of the present disclosure include pharmaceutical compositions for improving time to progression (TTP) in patients diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered 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.

[0503] Aspects of the present disclosure include pharmaceutical compositions for improving time to response (TTR) in patients diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered 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.

[0504] Aspects of the present disclosure include pharmaceutical compositions for improving duration of objective response (DOR) in patients diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered according to a 28-day cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0505] Aspects of the present disclosure include pharmaceutical compositions for improving clinical benefit rate (CBR) in patients diagnosed with B-cell non-Hodgkin lymphoma, the pharmaceutical compositions comprising a therapeutically effective amount of TNB-486 administered according to a 28-day cycle, wherein the therapeutically effective amount of TNB-486 is from about 30 μg to about 30,000 μg.

[0506] In some aspects of the pharmaceutical compositions 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%.

[0507] In some aspects of the pharmaceutical compositions for use, the treatment cycle is modified to increase more time between doses. In some aspects, the treatment cycle is modified by consistently eliminating one or more treatment cycles from the dosing regimen.

[0508] In some aspects of the pharmaceutical composition for use, the treatment cycle is modified to include a priming dose. In some aspects, the priming dose is from about 150 μg to about 1500 μg. In some aspects, the priming dose is from about 270 μg to about 1000 μg. In some aspects, the priming dose is administered at a first time point in the first treatment cycle, and the full dose is administered at all subsequent time points. In some aspects, 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 day 1 and day 15 of all subsequent treatment cycles.

[0509] In some aspects of the pharmaceutical composition for use, the treatment cycle is modified to include at least two priming doses. In some aspects, the first priming dose is from about 150 μg to about 540 μg. In some aspects, the second priming dose is from about 800 μg to about 1200 μg. In some aspects, the first priming dose is about 270 μg and the second priming dose is about 1000 μg. In some aspects, 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, the full dose is administered on day 15 of the first treatment cycle, and the full dose is administered on day 1 and day 15 of all subsequent treatment cycles.

[0510] In some aspects of the pharmaceutical composition for use, the full dose is equal to 100% of the full dose corresponding to the next lowest dose group or is more than 100% less than it. In some aspects, the full dose is equal to 50% of the full dose corresponding to the next lowest dose group or is more than 50% less than it. In some aspects, the full dose is equal to 33% of the full dose corresponding to the next lowest dose group or is more than 33% less than it.

[0511] In some aspects of the pharmaceutical composition for use, prior to administering TNB-486, the patient is premedicated with an agent that reduces the risk or severity of hypersensitivity. In some aspects, the agent that reduces the risk or severity of hypersensitivity is selected from the group consisting of dexamethasone, diphenhydramine, acetaminophen, ranitidine, tocilizumab, any equivalents thereof, or any combination thereof. In some aspects, the agent that reduces the risk or severity of hypersensitivity is administered from 15 minutes to 60 minutes prior to administering TNB-486. In some aspects 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.

[0512] ​

[0513] Results of a Phase 1 Study of TNB-486 in Patients with Relapsed / Refractory B-NHL

[0514] Background :

[0515] 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 silent IgG4 backbone confers a long half-life suitable for intermittent dosing. In DLBCL, the combination of CD19-targeting TNB-486 with CD20-targeting R-CHOP incorporated by rituximab (Rituxan) can result in synergistic tumor killing and reduce the risk of antigen escape, thus providing a promising approach for improving long-term remission. Here, the interim results of the first-in-human (FIH) phase 1 study of TNB-486 in R / R 8-NHL are presented.

[0516] Methods :

[0517] The primary objectives of the 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 R / R 8-NHL after at least 2 prior lines of therapy were eligible; prior anti-CD19 therapy was permitted. Patients received escalating doses of TNB-486 by IV infusion over 1 to 2 hours Q2W until PD / unacceptable toxicity occurred. Fixed doses were initially given at lower doses, followed by priming doses at higher target doses (>2.4 mg). Responses were evaluated by RECIL 2017, and adverse events were graded using CTCAE, except for cytokine release syndrome (CRS) and neurological toxicity (NT) graded according to ASTCT criteria.

[0518] Results :

[0519] As part of the study up to the first clinical cutoff, 27 subjects had received TNB-486 at doses ranging from 0.030 mg to 10 mg. Efficacy was evaluable in 22 patients and safety in 27 patients. Patient characteristics are summarized in Table 18. Overall, patients had received heavy pretreatment, with a median of 4 prior lines of therapy (range 2 - 21 prior lines), and 19% of enrolled subjects had prior CAR-T failure.

[0520] Enrollment was expanded, and the second clinical cutoff was evaluated in 30 patients, with efficacy evaluable in 25 patients and safety in 30 patients. Patient characteristics are summarized in Table 19. For the second clinical cutoff, 23% of enrolled subjects had prior CAR-T failure.

[0521] Summary of Enrolled Patients at the First Clinical Cutoff, Table 18

[0522]

[0523]

[0524] Summary of Enrolled Patients at the Second Clinical Cutoff, Table 19

[0525]

[0526] For the first clinical cut-off value, only one case of grade 3 CRS was observed and no CRS (any grade) occurred after cycle 1. The median time to onset of CRS was 1 day and the median time to resolution was 1.5 days (range <1 - 9). Eight subjects received tocilizumab; six of these subjects had grade 2 CRS. Six subjects (22%) experienced NT (7% grade 3, no grade 4, occurring in two subjects with MZL and Richter 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 after cycle 1. Four subjects received steroids for NT. Notably, one subject who experienced grade 3 NT continued treatment at a reduced dose without recurrence.

[0527] The CRS results were very similar at the second clinical cut-off value, as shown in Table 20 and Figure 5A as shown.

[0528] Summary of CRS Events at the Second Clinical Cutoff, Table 20

[0529] N＝30 Patients Who Experienced CRS 18(60％) Grade 1 11(37％) Grade 2 6(20％) Grade 3 1(3％) Onset, Median (Range) 1(0-2) Duration, Median (Range) 2(0-9) Tocilizumab for CRS 9(30％) Resolution 18(100％)

[0530] Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS) was evaluated at the second clinical cut-off value and the results are shown in Table 21 and Figure 5B as follows. 10 / 30 subjects (33%) reported events classified as neurotoxicity. All of these events resolved. Grade 3 events occurred in 4 patients with risk factors (e.g., age >80 years, aggressive histology and / or existing G4 events after CART). Three of these 4 patients were subsequently re-challenged without recurrence.

[0531] Summary of ICANS Events at the Second Clinical Cutoff, Table 21

[0532]

[0533]

[0534] Preliminary PK data showed an average T ranging from 7.55 days to 11.7 days at the active dose 1 / 2, thus supporting Q2W (or less frequent) dosing of TNB-486.

[0535] First clinical cutoff: At the first clinical cutoff date, the overall response rate (ORR) at 800 μg was 72% (13 / 18 evaluable subjects), with a CR rate of 61% (11 / 18). Among the CAR-T exposed subjects with evaluable responses, 2 / 3 achieved CR. Among the FL subjects with evaluable responses treated at 800 μg, the ORR was 88% (7 / 8 subjects); all responding subjects achieved CR. Five DLBCL subjects were treated at 800 μg, with an ORR of 40%; one subject achieved MRD-negative CR. Notably, this subject had previously been treated with 5 lines of therapy including CAR-T and had never achieved CR prior to this study. Among 4 subjects with MZL, the ORR was 75% (all CR). At a median follow-up of 3.8 months (range: 1.5 months to 8.7 months), no CR subjects relapsed, and remission was sustained up to 10 months after initiation of TNB-486 therapy ( Figure 6 ). Responses were observed across all NHL subtypes and prognostic factors (e.g., disease burden, existing lines of therapy, and refractoriness to existing therapies).

[0536] Second clinical cutoff: At the second clinical cutoff date, the overall response rate (ORR) was greater than 75% (19 / 25 evaluable subjects), with a CR rate of 63% (16 / 25). Among the CAR-T exposed subjects with evaluable responses, 2 / 4 achieved CR. Among the FL subjects with evaluable responses, the ORR was 88% (7 / 8 subjects), and all responding subjects achieved CR. See Figure 7 . At a median follow-up of 3.8 months (range: 1.5 months to 8.7 months), no CR subjects relapsed, and remission was sustained up to 10 months after initiation of TNB-486 therapy ( Figure 8 ). Responses were observed across all NHL subtypes and prognostic factors (e.g., disease burden, existing lines of therapy, and refractoriness to existing therapies). Preliminary data from the current FIH study of TNB-486 showed a tolerable safety profile, mainly with low-grade CRS / NT at doses up to 7.2 mg (dose escalation ongoing) and promising activity in heavily pretreated B-NHL.

[0537] Third Clinical Cutoff: At the third clinical cutoff date, 17 patients received TNB-486 at target doses of 0.03 mg - 10 mg (median age 68 years [range 33 - 86]; 53% male; 65% stage III / IV; 25% CD20 disease; median number of existing treatment lines [LOT] 3 [range 2 - 9]). Existing treatments included αCD20Ab (100%), alkylating agents (76%), IMiD (47%), CD20 TCE (12%), CD19 CAR T (12%), and ASCT (6%); within 24 months (POD24) of starting the 1st LOT, 53% progressed or started the 2nd LOT. The median study duration was 7 months (range 1 - 22). Eleven patients were evaluable for efficacy at target doses ≥2.4 mg. The objective response rate (ORR) and complete response (CR) rate were 91%, as Figure 10 shown. The ORR / CR for patients with CD20 disease, existing CD20 TCE, and POD24 was 100%. For the patient with CR, 1 patient with 6 existing LOTs progressed at C6 with retained 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%, including 1 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 >10% of patients included lymphopenia (35%) and neutropenia (12%).

[0538] Example 2

[0539] Subsequent Results of the Phase I Study

[0540] This example provides additional data obtained during Part 1, Group A of the Phase I study described in Example 1 above. A summary of the cohorts in Group A is provided in Table 22.

[0541] Summary of Group A Cohorts, Table 22

[0542]

[0543] For each cohort, overall response, objective response rate, and clinical benefit rate were evaluated as described above. A summary of the responses for each cohort is provided in Table 23. Abbreviations are defined as above.

[0544] Summary of Responses in Each Cohort, Table 23

[0545]

[0546] [1] The best overall response is defined as the best response observed for each subject after treatment and prior to initiation of any new anti-cancer therapy.

[0547] [2] The ORR is defined as the proportion of subjects with a confirmed partial or complete response to treatment as determined by RECIL 2017.

[0548] [3] The CBR is defined as the proportion of subjects with a confirmed CR, PR, or MR or SD at least 24 weeks after response to treatment as determined by RECIL 2017.

[0549] A summary of the responses of all subjects in all fixed and single-priming dose cohorts, all double-escalating priming dose cohorts, and combinations is provided in Table 24. Abbreviations are as defined above and are the same as in Table 23.

[0550] Combined Summary of Responses, Table 24

[0551]

[0552] [1] Confidence intervals are based on Clopper-Pearson confidence limits.

[0553] Outcomes in each cohort were also determined based on the subtype of B-cell non-Hodgkin lymphoma (B-NHL). Results for all fixed and single-priming dose cohorts of B-NHL subtypes are summarized in Table 25. Abbreviations used in the table below 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.

[0554] All Fixed and Single-Eliciting Cohorts of B-NHL Subtypes, Table 25

[0555]

[0556] Results by subtype for double-escalating priming dose cohorts 5d and 6d are shown in Tables 26 and 27.

[0557] Results of Cohort 5d (2400 ug Dose) of B-NHL Subtypes, Table 26

[0558]

[0559] Results of Cohort 6d (7200 ug Dose) of B-NHL Subtypes, Table 27

[0560]

[0561] Target doses of 2400 μg or greater were found to have significant ORR and CBR. A summary of the results by B-NHL subtype for all doses 2400 μg or greater is provided in Table 28.

[0562] Results of Doses 2400 ug or Greater of B-NHL Subtypes, Table 28

[0563]

[0564] Measure cytokine release of the following cytokines after dose administration: interleukin - IL - 2, IL - 6, IL - 8, and IL - 10; interferon γ (INFγ); monocyte chemoattractant protein - 1 (MCP - 1); macrophage inflammatory protein - 1α (MIP - 1α); tumor necrosis factor α (TNFα). In any of the groups, no cytokine release of IL - 2, IL - 6, MIP - 1α, INFγ, and TNFα was observed. Some release of IL - 8 and IL - 10 was observed, which generally subsided after 9 to 24 hours. Release of MCP - 1 was observed in all groups, but for most doses, it also subsided after 9 to 24 hours.

Claims

1. A method for treating B-cell non-Hodgkin lymphoma (B-NHL) in a patient in need thereof, 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, optionally wherein the patient receives six treatment cycles.

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

3. The method according to any one of the preceding claims, wherein TNB-486 is administered to the patient as a single therapy.

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

5. The method according to any one of the preceding claims, wherein the patient has received at least two prior lines of systemic therapy.

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

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

8. The method according to any one of the preceding claims, wherein the patient has sufficient bone marrow function.

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

10. The method according to any one of the preceding claims, wherein the patient has: total bilirubin less than or equal to 1.5 times the upper limit of normal; aspartate aminotransferase (AST) less than or equal to 3 times the upper limit of normal; and alanine aminotransferase (ALT) less than or equal to 3 times the upper limit of normal.

11. A method for improving the objective response rate (ORR) in a patient diagnosed with B-cell non-Hodgkin 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.

12. A method for improving the overall survival (OS) rate in a patient diagnosed with B-cell non-Hodgkin 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.

13. A method for improving the progression-free survival (PFS) rate in a patient diagnosed with B-cell non-Hodgkin 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.

14. A method for improving the time to progression (TTP) in a patient diagnosed with B-cell non-Hodgkin 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.

15. A method for improving the time to response (TTR) in a patient diagnosed with B-cell non-Hodgkin 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.

16. A method for improving the duration of objective response (DOR) in a patient diagnosed with B-cell non-Hodgkin 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.

17. A method for improving the clinical benefit rate (CBR) in a patient diagnosed with B-cell non-Hodgkin 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.

18. The method according to 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. The method according to any one of the preceding claims, wherein the treatment cycle is modified to increase the time between doses.

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

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

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

24. The method according to any one of claims 21 to 22, wherein the priming dose is administered at a first time point in the first treatment cycle and the full dose is administered at all subsequent time points.

25. The method according to 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 day 1 and day 15 of all subsequent treatment cycles.

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

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

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

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

30. The method according to any one of claims 26 to 29, wherein 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, 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.

31. The method according to claim 24, 25 or 30, wherein the full dose is equal to 100% of the full dose corresponding to the next lowest dose group or is more than 100% less than it.

32. The method according to claim 24, 25 or 30, wherein the full dose is equal to 50% of the full dose corresponding to the next lowest dose group or is more than 50% less than it.

33. The method according to claim 24, 25 or 30, wherein the full dose is equal to 33% of the full dose corresponding to the next lowest dose group or is more than 33% less than it.

34. The method according to any one of the preceding claims, the method further comprising premedicating the patient with an agent that reduces the risk or severity of hypersensitivity prior to administration of TNB-486.

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

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

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

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

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

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

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

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

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

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

47. The method according to any one of the preceding claims, wherein TNB-486 is a bispecific molecule that binds to CD3 and human CD19, the bispecific molecule comprising: (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 portion that binds to human CD3; and (iii) a third polypeptide subunit that binds to human CD19 and comprises the amino acid sequence of SEQ ID NO:20.

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