Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2815—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/35—Valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/515—Complete light chain, i.e. VL + CL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site

Definitions

• TCEs CD3-based bispecific T cell engagers
• TCEs simultaneously bind a tumor associated antigen (TAA) and cluster of differentiation 3 (CD3) on a T cell to form a T cell receptor (TCR)-independent artificial immune synapse, circumventing human leukocyte antigen (HLA) restriction, and inducing T cell activation and cytolysis of the tumor cell.
• TAA tumor associated antigen
• CD3 cluster of differentiation 3
• HLA human leukocyte antigen
• the first generation of TCEs were simple bispecific T cell engagers (BiTEs), composed of two tandem single-chain variable fragments (scFvs) including a strong CD3 -binding arm and a TAA binding domain.
• BiTEs simple bispecific T cell engagers
• scFvs tandem single-chain variable fragments
• CD3 using the Orthoclone 0KT3 antibody
• CD 19 cluster of differentiation 19
• BiTE formats exhibit very short half-life and have poor manufacturability (Ellerman, "Bispecific T cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety,” Methods 154: 102-17 (2019)).
• the second generation of TCEs include a fragment crystallizable (Fc) domain which can be modified to confer half-life extension and mutations to eliminate Fc receptor (FcR) binding, and present improved manufacturability (Vafa et al., "Perspective: Designing T cell Engagers With Better Therapeutic Windows,” Front Oncol. 10: 446 (2020)). Nevertheless, those molecules still include high affinity-CD3 binding domains, linking to induction of neurotoxicity and CRS in the clinic.
• Fc fragment crystallizable domain
• both CD3-based BiTEs and novel immunoglobulin G (IgG)-format TCEs bind and activate both cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) T cells, potentially engaging unfavorable T cells such as regulatory T cells (Treg), which have been shown to potentially decrease the cytolytic activity of CD8 T cells (Duell et al., "Frequency of regulatory T cells determines the outcome of the T cell-engaging antibody blinatumomab in patients with B-precursor ALL," Leukemia 31 : 2181-90 (2017)).
• CD4 cluster of differentiation 4
• CD8 T cells regulatory T cells
• the disclosure provides a binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the formula: VL-CL and a third polypeptide chain has a structure represented by the formula: VHI-CHI-VH2-FC and a fourth polypeptide chain has a structure represented by the formula: VHI-CHI-VH3-FC wherein: VL is an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen; VHI is an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen; CL is an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen; CHI is an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor- associated antigen; VH2 is a heavy chain variable domain that specifically binds a T cell receptor; VH3 is
• a binding protein comprising comprising three polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the first polypeptide chain has a structure represented by the formula: VL-CL; and a second polypeptide chain has a structure represented by the formula:VHi-CHi-VH2-Fc; and a third polypeptide chain has a structure represented by the formula: VHI-VH3-FC; wherein: VL is an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen; VHI is an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen; CL is an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen; CHI is an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor-associated antigen; VH2 is a heavy chain variable domain that specifically binds a T
• a binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co- stimulatory molecule binding site, wherein the polypeptide chains comprise (a) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; (b) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4; (c) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 10; (d) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 12; (e) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 14; (f) the amino acid of sequences of SEQ ID NO: 1 and SEQ ID NO: 19; (g) the amino acid of sequences of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1,
• Fig. 1 illustrates a representative binding protein format of the present disclosure, including two binding sites to a tumor associated antigen (TAA), one binding site to T cell receptor (TCR) on T cells, and one binding site to a T cell costimulatory molecule.
• TAA tumor associated antigen
• TCR T cell receptor
• T cell costimulatory molecule Upon cleavage by tumor specific protease at the site indicated, the Fc domain is released, and T cells are activated through TCR binding and activation of a costimulatory receptor.
• Fig. 6 shows a line graph depicting percentage of surface CD25+ cells using Raji cells as a means of analyzing CD4 and CD8 T cell activation profiles.
• Figs. 7A-7C show in vitro cytolysis and T cell activation profiles induced by CD20 T cell MCAZ 88 binding protein.
• Fig. 7A shows a representative structure of the MCAZ 88 binding protein.
• Fig. 7B shows that MCAZ 88 induced cytolysis assessed on Raji cell line (CD20+, 85371 antigen per cell).
• Fig. 7C shows CD4 and CD8 T cell activation profiles assessed by measuring the % of surface CD25+ cells.
• Figs 9A-9B show bar graphs illustrating non-specific CD8 and CD4 T cell activation in plate-adsorbed antibodies as assessed by flow cytometry detecting CD25+ surface expression.
• Fig. 9A shows non-specific CD8 activation. Binding proteins as shown from top to bottom in the legend are depicted from left to right along the x axis.
• Fig. 9B shows non-specific CD4 activation.
• Figs 13A-13E show evaluation of TCR VHH and CD8 VHH bispecific binding proteins.
• Fig. 13A shows the structure of the MCAZ 8.69 binding protein.
• Fig. 13B shows the structure of the MCAZ 8.70 binding protein.
• Fig. 13C shows cytolysis assessed on CD20+ B Raji cell line (expressing 85000 CD20 antigen per cell).
• Fig. 13D shows cytolysis EC50 values for each cell line.
• Fig. 13E shows CD4 and CD8 T cell activation profiles assessed as %CD25 expressing cells.
• Figs. 14A-14B show non-specific T cell activation assessment for various binding proteins. Different concentrations of binding proteins were plate-bound and 1.5e5 purified T cells were added, in the absence of tumor cells and associated proteases. After 48h incubation, unspecific (Fig. 14A) CD8 and (Fig. 14B) CD4 T cell activation levels were assessed by flow cytometry as %CD69+/CD25+ surface expression.
• FIG. 21 shows PK assessment after a single dose of the MCAZ 7.1 binding protein.
• NSG mice humanized with panT cells were injected with 0.5 mg/kg of binding protein as indicated and systemic concentrations of binding proteins were measured after Ih, 6h, 24h, 48h, 72h, and 168h.
• Figs. 23 A-23F show in vivo cytokine release assessment of the MCAZ 7.1 binding protein compared to the CD3xCD20 bivalent engager in a CRS mouse model. Mean +/- SD are plotted for (Fig. 23A) IL-6, (Fig. 23B) TNF-a, (Fig. 23C) IL-10, (Fig. 23D) IFN-g, (Fig. 23E) IL-2, and (Fig. 23F) IL- 17 A.
• Figs. 29A-29B show in vitro cytotoxicity and T cell activation bias for MDA-MB-231- EGFR high.
• Fig 29 A shows equivalent cytolytic activities of EGFR TITAN and broad CD3 engager used as a comparator on MDA-MB-231 tumor cell line.
• Fig. 29B shows strong biased CD8 T cell activation profile for EGFR TITAN (MCAZ13.8) as highlighted by the %CD25 surface expression on T cells.
• Figs. 30A-30E show that EGFR TITAN (MCAZ13.8) selective engagement of CD8 T cells during cytolysis is associated with significantly lower cytokine release than broad CD3+ T cell engagement by CD3xEGFR bivalent comparator.
• Figs. 31 A- 3 IB show T cell activation.
• Fig. 31 A shows that EGFR TITAN (MCAZ 13.8) binding protein does not activate T cells when plate-bound at different concentrations.
• the broad CD3 engager used as a comparator induces significant T cell activation at the highest concentrations used.
• Fig. 3 IB shows that the absence of T cell activation induced by plate-bound EGFR TITAN (MCAZ 13.8) is associated with absence of cytokine release.
• ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. For example, “about 5%” means “about 5%” and also “5%.” The term “about” can also refer to ⁇ 10% of a given value or range of values. Therefore, about 5% also means 4.5%-5.5%, for example.
• variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.
• the variable regions of each light/heavy chain pair typically form an antigen binding site.
• the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
• the CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope.
• both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
• Antigen-binding fragment thereof refer to at least the minimal portion of an antibody which is capable of binding to a specified antigen which the antibody targets, e.g., at least some of the complementarity determining regions (CDRs) of the variable domain of a heavy chain (VH) and the variable domain of a light chain (VL) in the context of a typical antibody produced by a B cell.
• CDRs complementarity determining regions
• the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2).
• class e.g., IgG, IgA, and IgE
• subclass e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2
• native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
• native Fc is generic to the monomeric, dimeric, and multimeric forms.
• the term "antigen" or "target antigen,” as used herein, refers to a molecule or a portion of a molecule that is capable of being recognized by and bound by the antigen binding portion of the binding proteins of the disclosure.
• the target antigen is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
• a target antigen may have one or more epitopes. With respect to each target antigen recognized by the antigen binding portion of the binding protein, is capable of competing with an intact antibody that recognizes the target antigen.
• antigen binding site refers to a site created on the surface of a binding protein of the disclosure where an antigen or an epitope on an antigen is bound.
• nucleotide includes a singular nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
• mRNA messenger RNA
• pDNA plasmid DNA
• nucleic acid includes any nucleic acid type, such as DNA or RNA.
• treating refers to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival rates, and/or reducing discomfort.
• treating can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
• Administration refers to providing, contacting, and/or delivering a binding protein by any appropriate route to achieve the desired effect.
• Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.
• the terms "subject,” “individual,” or “patient,” refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
• Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
• composition refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.
• the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of binding proteins of the disclosure.
• pharmaceutically acceptable carrier or “physiologically acceptable carrier,” as used herein, refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of one or more binding proteins of the disclosure.
• the binding proteins disclosed herein may be formulated with a pharmaceutically acceptable carrier, excipient, or stabilizer, as pharmaceutical compositions.
• compositions are suitable for administration to a human or non-human animal via any one or more routes of administration using methods known in the art.
• pharmaceutically acceptable carrier means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
• Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
• Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
• contemplated carriers, excipients, and/or additives which may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium, and the like.
• These and additional known pharmaceutical carriers, excipients, and/or additives suitable for use in the formulations described herein are known in the art, for example, as listed in “Remington: The Science & Practice of Pharmacy,” 21st ed., Lippincott Williams & Wilkins, (2005), and in the “Physician's Desk Reference,” 60th ed., Medical Economics, Montvale, N.J. (2005).
• Pharmaceutically acceptable carriers can be selected that are suitable for the mode of administration, solubility, and/or stability desired or required.
• a binding protein comprising one T cell costimulatory molecule binding site.
• a co-stimulatory molecule comprises a co-stimulatory domain capable of potentiating or modulating the response of immune effector cells.
• Co-stimulatory domains can include sequences, for example, from one or more of CD3zeta (or CD3z), CD28, CD137 (4- 1BB), OX-40, ICOS, CD27, GITR, CD2, IL-2RP and MyD88/CD40.
• the T cell costimulatory molecule is CD8.
• the T cell costimulatory molecule is CD137 (4- 1BB).
• a binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the first and second polypeptide chains have a structure represented by the formula: VL-CL; and a third polypeptide chain has a structure represented by the formula: VHI-CHI-VH2-FC; and a fourth polypeptide chain has a structure represented by the formula: VHI-CHI-VH3-FC; wherein VL is an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen;Vm is an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen; CL is an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen; CHI is an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor- associated antigen; VH2 is a heavy chain variable domain that specifically binds a T
• VL-CL an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen
• VHI an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen
• CL an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen
• CHI an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor-associated antigen
• VH2 is a heavy chain variable domain that specifically binds a T cell receptor
• Fc is an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain
• VL-CL an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen
• VHI an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen
• CL an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen
• CHI an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor-associated antigen
• VH2 is a heavy chain variable domain that specifically binds a T cell co-stimulatory molecule
• VH3 an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen
• VHI an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen
• CL an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen
• CHI an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor-associated antigen
• VH2 is a heavy chain variable domain that specifically binds a T cell co-
• a binding protein comprising comprising three polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the first polypeptide chain has a structure represented by the formula: VL-CL; and a second polypeptide chain has a structure represented by the formula:VHi-CHi-VH2-Fc; and a third polypeptide chain has a structure represented by the formula: VHI-VH3-FC; wherein: VL is an immunoglobulin light chain variable domain that specifically binds a tumor-associated antigen; VHI is an immunoglobulin heavy chain variable domain that specifically binds a tumor-associated antigen; CL is an immunoglobulin light chain constant domain that specifically binds a tumor-associated antigen; CHI is an immunoglobulin CHI heavy chain constant domain that specifically binds a tumor- associated antigen; VH2 is a heavy chain variable domain that specifically binds a
• the heavy chain variable domain that specifically binds a T cell co- stimulatory molecule is an immunogloblulin heavy chain variable domain.
• the heavy chain variable domain that specifically binds a T cell co-stimulatory molecule is a single domain sequence.
• the heavy chain variable domain that specifically binds a T cell co-stimulatory molecule is a nanobody.
• the heavy chain variable domain that specifically binds a T cell co-stimulatory molecule is a camelid.
• the heavy chain variable domain that specifically binds a T cell co-stimulatory molecule is a single-domain variable new antigen receptor.
• the heavy chain variable domain that specifically binds a T cell receptor binding site is an immunogloblulin heavy chain variable domain. In some aspects, the heavy chain variable domain that specifically binds a T cell receptor binding site is a single domain sequence. In particular aspects, the heavy chain variable domain that specifically binds a T cell receptor binding site is a nanobody. In particular aspects, the heavy chain variable domain that specifically binds a T cell receptor binding site is a camelid. In particular aspects, the heavy chain variable domain that specifically binds a T cell receptor binding site is a single-domain variable new antigen receptor.
• the Fc of the binding protein is from an IgG antibody for example IgGl, IgG2, IgG3, IgAl, and IgGA2.
• the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 47). In some aspects, the linker comprises the amino acid sequence AAAYPYDVPDYGSGEGTSTGSGGSGGSGGA (SEQ ID NO: 48). In some aspects, the linker further comprises a hemagglutinin tag.
• the binding protein comprises Hi, an immunoglobulin hinge region positioned between CHI and VH2 on the third polypeptide chain and H2, an immunoglobulin hinge region positioned between VH2 and the Fc on the third polypeptide chain, wherein Hi and H2 are each independently an immunoglobulin hinge region or are absent.
• the binding protein compiles H3, an immunoglobulin hinge region positioned between CHI and VH3 on the fourth polypeptide chain and H4, an immunoglobulin hinge region positioned between VH3 and the Fc on the fourth polypeptide chain, wherein H3 and H4 are each independently an immunoglobulin hinge region or are absent.
• HI,H2, H3, and H4 are each independently an immunoglobulin hinge region or are absent.
• the binding protein comprises a first and a second polypeptide chain having a structure represented by the formula:
• VL-CL VL-CL and a third polypeptide chain having a structure represented by the formula:
• VHI-CHI-HI-LI-VH2-H2-L2-FC VHI-CHI-HI-LI-VH2-H2-L2-FC and a fourth polypeptide chain having a structure represented by the formula:
• the binding protein comprises a first polypeptide chain having a structure represented by the formula:
• VL-CL VL-CL and a second polypeptide chain having a structure represented by the formula:
• VHI-CHI-HI-LI-VH2-H2-L 2 -FC VHI-CHI-HI-LI-VH2-H2-L 2 -FC and a third polypeptide chain having a structure represented by the formula:
• the binding protein comprises two polypeptide chains having a structure represented by the formula:
• III-VL-CL-IF III-VL-CL-IF; and two polypeptide chains having a structure represented by the formula:
• the binding protein comprises a first polypeptide chain having a structure represented by the formula:
• VL-CL VL-CL and a second polypeptide chain having a structure represented by the formula:
• VHI-CHI-VH2-FC and a third polypeptide chain having a structure represented by the formula:
• the binding protein comprises four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the polypeptide chains comprise
• the binding protein comprises three or four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise
• a binding protein comprising three polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell co-stimulatory molecule binding site, wherein the polypeptide chains comprise amino acid sequences having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-56.
• Some aspects of the present disclosure relate to an isolated nucleic acid sequence encoding a binding protein as described herein.
• the isolated nucleic acid sequence may be included in a vector.
• the activation of T cells through methods of the present disclosure can be determined by measuring the percentage of surface interleukin-2 receptor alpha chain-positive (CD25+) T cells.
• the percentage of surface CD25+ T cells that are CD8 T cells can be higher than the percentage of surface CD25+ T cells that are CD4 T cells.
• activation of T cells can be determined by the percentage of CD69+/CD25+ T cells.
• activation of T cells can be determined by measuring levels of cytokines released by activated T cells.
• the method of treatment of the present disclosure can result in reduced engagement of regulatory T cells (Tregs), increased cytolytic activity, and/or reduced incidence of cytokine release syndrome (CRS) relative to that resulting from bispecific T-cell engager (BiTEs) previously known in the art.
• the disclosure also provides a therapeutically effective amount of a binding protein for use in reducing engagement of regulatory T cells (Tregs), increasing cytolytic activity, and/or reducing incidence of cytokine release syndrome (CRS) relative to that resulting from bispecific T-cell engager (BiTEs) previously known in the art.
• the inflammatory disease or inflammatory disease where B cells are involved is selected from rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, pemphigus, lupus profundus, sceleroderma, discoid lupus erythematosus, systemic lupus erythematosus, Sjogren’s syndrome, atopic dermatitis and allergic contact dermatitis.
• MCAZ 88, MCAZ 7.5, MCAZ 89 were each generated with a unique placement of the TCR binding domain or absence of the TCR binding domain.
• MCAZ 7.5 had the TCR binding domain placed in the hinge portion of the binding protein ( Figure 8 A)
• Modified linker lengths were tested to determine the optimal linker for stability and function of the binding protein.
• MCAZ 7.7 was generated with a modified longer linker (GGGGSGGGGS) positioned between the CD20 binding domain and the TCR-binding domain ( Figure 11 A) as compared to MCAZ 7.1 that had a linker (T) positioned between the CD20 binding domain and the TCR-binding domain.
• MCAZ 10.1 was generated with a more rigid CD8 arm as compared to MCAZ 7.1.
• MCAZ 10.1 was generated with the linker positioned between the CD20 binding domain and the TCR-binding domain deleted as compared to MCAZ 7.1 that had a linker (T) and a shortened linker (G) positioned between the TCR-binding domain and the Fc as compared to MCAZ 7.1 that had a linker (GEGTSTGSGGSGGSGGA (SEQ ID NO: 49)).
• T linker
• G shortened linker
• MCAZ 7.7 cytolysis was assessed on CD20+ Raji B cell line (expressing 85000 CD20 antigen per cell). B cell lines were CTV stained and incubated PBMCs at an E:T ratio of 5: 1 for 4 days. % cytolysis was measured by flow cytometry.
• a 3D-spheroid model was used to determine cytolytic activity of the MCAZ 7.1 variant as compared to the CD3xCD20 bivalent engager.
• GFP-expressing CD20+ B cell line (TMD8, 100 000 CD20/cell) were plated in low adherent plate to form 3D-spheroids for 72 h. Then, purified panT cells were added at a 15: 1 E:T ratio and co-incubated with no engager, TENG0093, or CD3xCD20 bivalent engager for 96h.
• GFP+ TMD8 cells were quantified using a Cellinsight CX7 HCS Platform imager.
• CD8-specifc engagement of MCAZ7.1 provided similar Emax killing of CD20+ tumor cells as the CD3xCD20 bivalent engager, but cytolysis was associated with significantly lower pro-inflammatory cytokine release than the CD3+ T cell engagement by the CD3xCD20 bivalent engager.
• binding protein Based on the multitude of binding protein formats tested, a binding protein was identified with a novel conformation comprising the TCR binding domain and the T-cell co-stimulatory domain on separate arms located at the hinge region using an IgG Fc.
• This binding protein advantageously had increased cytolytic activity and reduced incidence of cytokine release both in vitro and in vivo in the absence of tumor target cells (non-specific T cell activation) and during cytolytic activity in the presence of tumor target cells.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Hematology (AREA)
• Oncology (AREA)
• Peptides Or Proteins (AREA)
• Medicinal Preparation (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=86226585

Family Applications (1)

Country Status (5)

Cited By (4)

Citations (7)

• 2023
  • 2023-04-06 JP JP2024559145A patent/JP2025512953A/ja active Pending
  • 2023-04-06 US US18/854,215 patent/US20250346681A1/en active Pending
  • 2023-04-06 CN CN202380032014.9A patent/CN118974091A/zh active Pending
  • 2023-04-06 WO PCT/EP2023/059278 patent/WO2023198635A1/en not_active Ceased
  • 2023-04-06 EP EP23719654.8A patent/EP4508080A1/en active Pending

Patent Citations (7)

Non-Patent Citations (9)

Also Published As

Similar Documents

Legal Events