Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
  • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57434—Specifically defined cancers of prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • 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
  • C07K2317/50—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
  • C07K2317/565—Complementarity determining region [CDR]
• • 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
  • C07K2317/70—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
  • C07K2317/77—Internalization into the cell
• • 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
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the invention relates to prostate specific membrane antigen (PSMA) binding molecules, and the use of such binding molecules in the treatment of disease.
• PSMA prostate specific membrane antigen
• Prostate cancer is the most commonly diagnosed non-skin-related malignancy in males in developed countries. It is estimated that one in six males will be diagnosed with prostate cancer.
• prostate cancer has greatly improved following the use of serum-based markers such as the prostate specific antigen (PSA).
• PSA prostate specific antigen
• prostate tumour-associated antigens offer targets for tumour imaging, diagnosis, and targeted therapies.
• PSMA prostate specific membrane antigen
• a prostate tumour associated marker is such a target.
• PSMA is a 750-residue type II transmembrane glycoprotein highly restricted to prostate secretory epithelial cell membranes. It is highly expressed in prostate cancer cells and in nonprostatic solid tumor neovasculature and expressed at lower levels in other tissues, including healthy prostate, kidney, liver, small intestine, and brain. PSMA expression increases with prostate disease progression and metastasis and its expression level has thus been correlated with tumour aggressiveness. Various immunohistological studies have demonstrated increased PSMA levels in virtually all cases of prostatic carcinoma compared to those levels in benign prostate epithelial cells.
• PSMA staining is found in all stages of the disease, including prostatic intraepithelial neoplasia, late stage androgen-independent prostate cancer and secondary prostate tumours localized to lymph nodes, bone, soft tissue, and lungs. PSMA is thus widely used as a biomarker for prostate cancer cells.
• PSMA has a 3-part structure: a 19-amino-acid internal portion, a 24-amino-acid transmembrane portion, and a 707-amino-acid external portion. It forms a noncovalent homodimer that possesses glutamate carboxypeptidase activity based on its ability to process the neuropeptide N-acetylaspartylglutamate and glutamate-conjugated folate derivatives. PSMA is rapidly and efficiently internalized by an endocytic pathway and rapidly recycles back to the membrane.
• Antibody-based therapeutics have emerged as important components of therapies for an increasing number of human malignancies in such fields as oncology, inflammatory and infectious diseases. In most cases, the basis of the therapeutic function is the high degree of specificity and affinity the antibody-based drug has for its target antigen. Arming monoclonal antibodies (mAbs) with drugs, toxins, or radionuclides is yet another strategy by which mAbs may induce a therapeutic effect. By combining the targeting specificity of an antibody with the tumour killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue thereby resulting in fewer side effects than most conventional chemotherapeutic drugs.
• mAbs monoclonal antibodies
• mAbs Due to their size and other physical properties, however, mAbs have to be administered either intravenously (iv) or subcutaneously (sc) and therefore have a high systemic exposure. Thus, their route of delivery can often be suboptimal, resulting either in antibody binding to target antigen at non-disease locations (potentially compromising the healthy function of normal, non-disease tissue) or resulting in suboptimal PK/PD characteristics. Either outcome may result in a loss of efficacy and/or a compromised safety profile by virtue of the suboptimal route of administration.
• this antibody recognizes an intracellular epitope of PSMA exposed upon cell death which limits its usefulness as an imaging agent for the detection of PSMA.
• mAbs such as J591 that recognize the extracellular portion of PSMA have been identified.
• the aim of the present invention is to address the need of alternative antibody-based treatments for use in the treatment of prostate cancer. Summary of the invention
• the invention relates to a binding molecule capable of binding human PSMA comprising a single human variable heavy chain domain (V H ) antibody selected from one of the following: a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 1 , a CDR2 sequence comprising SEQ ID NO. 2 and a CDR3 sequence comprising SEQ ID NO. 3; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 5, a CDR2 sequence comprising SEQ ID NO. 6 and a CDR3 sequence comprising SEQ ID NO. 7; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 9, a CDR2 sequence comprising SEQ ID NO.
• V H human variable heavy chain domain
• a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 13, a CDR2 sequence comprising SEQ ID NO. 14 and a CDR3 sequence comprising SEQ ID NO. 15; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 17, a CDR2 sequence comprising SEQ ID NO. 18 and a CDR3 sequence comprising SEQ ID NO. 19; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 21 , a CDR2 sequence comprising SEQ ID NO. 22 and a CDR3 sequence comprising SEQ ID NO.
• a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 25, a CDR2 sequence comprising SEQ ID NO. 26 and a CDR3 sequence comprising SEQ ID NO. 27; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 29, a CDR2 sequence comprising SEQ ID NO. 30 and a CDR3 sequence comprising SEQ ID NO. 31 ; a single domain V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 33, a CDR2 sequence comprising SEQ ID NO. 34 and a CDR3 sequence comprising SEQ ID NO. 35 or a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 37, a CDR2 sequence comprising SEQ ID NO. 38 and a CDR3 sequence comprising SEQ ID NO. 39.
• said CDR1 said CDR1 sequence comprises SEQ ID NO. 25
• said CDR2 sequence comprises SEQ ID NO. 26
• said CDR3 sequence comprises or consists of V H domain antibody 1.27 (SEQ ID NO. 28).
• said single domain V H domain antibody comprises or consists of a sequence selected from SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40.
• said single domain V H domain antibody comprises or consists of SEQ ID NO 28.
• said binding molecule comprises a first single human heavy chain variable immunoglobulin (V H ) domain antibody capable of binding human PSMA as described above and a second single V H domain antibody capable of binding human PSMA.
• V H variable immunoglobulin
• the invention relates to a pharmaceutical composition comprising a binding molecule as described above.
• the invention relates to an immunoconjugate comprising a binding molecule as described above. In one aspect, the invention relates to a method for treating prostate cancer or a prostatic disorder comprising administering a therapeutically-effective amount of a binding molecule, an immunoconjugate or a pharmaceutical composition as described above.
• the invention relates to a binding molecule, an immunoconjugate or a pharmaceutical composition as described above for use as medicament.
• the invention relates to a binding molecule, an immunoconjugate or a pharmaceutical as described above for use in the treatment of prostate cancer or a prostatic disorder.
• the invention relates to the use of a binding molecule, an immunoconjugate or a pharmaceutical composition according as described above in the manufacture of a medicament for the treatment of prostate cancer or a prostatic disorder.
• the invention relates an in vivo or in vitro method for reducing human PSMA activity comprising contacting human PSMA with a binding molecule as described above
• the invention relates method for determining the presence of PSMA in a test sample by an immunoassay comprising contacting said sample with a binding molecule as described above and at least one detectable label.
• the invention relates an isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding molecule as described above.
• the invention relates an immunoconjugate comprising a binding molecule as described above.
• the invention relates an immunoconjugate of the formula A-(L-D)n wherein A is an antigen-binding moiety comprising a first human single V H domain antibody capable of binding specifically to human PSMA as defined above, optionally comprising a second human single V H domain antibody capable of binding specifically to human PSMA and optionally comprising a third human single V H domain antibody, L is a linker, and D is an auristatin or a derivative thereof and n is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
• Figure 1 Binding of purified anti-PSMA V H in FMAT Mirrorball Assay. 1a #1.1 , -3.1 , A2.10, T2.1 , 1 b ⁇ 2.1 , ⁇ 2.13, T2.17 02.15, o2.12 ⁇ 2.22 1c single domain antibodies tested as shown by symbols from top to bottom ⁇ 1.8, ⁇ 1.10, A 1 .1 1 , T 1.12, 1 .13, o 1.14, 1.16, ⁇ 1 .17, 1.18.
• Figure 10 in vivo imaging 24h radiograph a) benchmark mAb, b) V H 2.1 , c) V H 2.1 -MSA, d) V H 1.1 , e) negative control.
• FIG. 11 Comparison of PSMA+ tumor, PSMA- tumor and blood biodistribution at 5min to 24h. a) V H 1.1 b) V H 2.1 c) V H 2.1 half life extended d) benchmark mAb e) HEL4 control.
• FIG. 13 Comparison of lungs, heart, liver, muscle and PSMA expressing (PSMA+) tumor biodistribution biodistribution at 5min to 24h. a) V H 1.1 b) V H 2.1 c) V H 2.1 half life extended d) benchmark mAb e) HEL4 control.
• Figure 16. shows in vitro cytotoxicity of monomeric MMAE-conjugated V H (A and B), bivalent V H (C and D) and biparatopic V H (E and F) on human cells stably expressing human PSMA protein and matched parental cells (PSMA negative) at a 48 hour incubation time point.
• Figure 17. shows in vitro cytotoxicity of MMAE-conjugated V H on human cells stably expressing human PSMA protein at a 72 hour incubation time point.
• ADC Pro_006 control antibody drug conjugates
• the invention provides isolated PSMA binding molecules, in particular those comprising at least one single V H domain antibody, that bind human PSMA, pharmaceutical compositions comprising such binding molecules, as well as isolated nucleic acids, recombinant expression vectors and isolated host cells for making such binding proteins and fragments. Also provided are methods of using the binding proteins disclosed herein to detect human PSMA, to inhibit human PSMA either in vitro or in vivo, and methods of treating disease.
• isolated human anti-human PSMA binding molecules specifically those comprising, or consisting of, single V H domain antibodies that bind to human PSMA with high affinity a slow off rate.
• the PSMA binding molecules of the invention bind to wild type human PSMA (Accession NO. Q04609).
• the sequence for the monomer is shown below (SEQ ID No. 250).
• the PSMA binding molecules of the invention bind to wild type human PSMA and/or cyno PSMA.
• PSMA binding molecule PSMA binding protein
• anti-PSMA single domain antibody or “anti-PSMA antibody” as used herein all refer to a molecule capable of binding to the human PSMA antigen.
• PSMA binding molecule includes a PSMA binding protein.
• the binding reaction may be shown by standard methods (qualitative assays) including, for example, a binding assay, competition assay or a bioassay for determining the inhibition of PSMA binding to its receptor or any kind of binding assays, with reference to a negative control test in which an antibody of unrelated specificity. Suitable assays are shown in the examples.
• An antibody or binding molecule of the invention including a single domain antibody and multivalent or multispecific binding agent described herein, "which binds" or is “capable of binding” an antigen of interest, e.g. PSMA, is one that binds, i.e. targets, the PSMA antigen with sufficient affinity such that it is useful in therapy in targeting a cell or tissue expressing the antigen.
• an antigen of interest e.g. PSMA
• Binding molecules of the invention bind specifically to human PSMA.
• binding to the PSMA antigen is measurably different from a non-specific interaction.
• the single domain antibodies of the invention bind to human PSMA and also bind to cyno PSMA.
• telomere binding or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10 "4 M, alternatively at least about 10 ⁇ 5 M, alternatively at least about 10 "6 M, alternatively at least about 10 ⁇ 7 M, alternatively at least about 10 ⁇ 8 M, alternatively at least about 10 ⁇ 9 M, alternatively at least about 10 ⁇ 10 M, alternatively at least about 10 ⁇ 11 M, alternatively at least about 10 "12 M, or greater.
• the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
• epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope).
• Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acids in a unique spatial conformation.
• Methods for determining what epitopes are bound by a given antibody or antibody fragment i.e., epitope mapping
• An antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes.
• the most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in different formats, using either labelled antigen or labelled antibody.
• the invention provides isolated PSMA binding molecules that bind human PSMA, pharmaceutical compositions and formulations comprising such binding molecule, as well as isolated nucleic acids encoding such binding molecules, recombinant expression vectors and host cells comprising such nucleic acids for making such binding molecules. Also provided by the invention are methods of using the binding molecules disclosed herein to detect human PSMA, to inhibit human PSMA either in vitro or in vivo, and methods of treating disease.
• isolated human anti-human PSMA binding molecules specifically those comprising, or consisting of, at least one single human V H domain antibody that binds to human PSMA with high affinity, a slow off rate.
• the invention relates to an isolated single variable domain antibody, an isolated variable single domain or an isolated immunoglobulin single variable domain wherein said isolated single domain antibody, isolated variable single domain or isolated immunoglobulin single variable domain binds to human PSMA.
• Binding molecules comprising at least one single domain antibody, variable single domain or immunoglobulin single variable domain are also within the scope of the invention. Fragments of the single domain antibody, variable single domain or immunoglobulin single variable domain that bind to human PSMA are also within the scope of the invention.
• Single domain antibody, variable single domain or immunoglobulin single variable domain are all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein.
• Single heavy chain variable domain antibodies do not comprise an immunoglobulin light chain.
• preferred embodiments of the various aspects of the invention relate to single heavy chain variable domain antibodies/immunoglobulin heavy chain single variable domains which bind a PSMA antigen in the absence of light chain.
• Human heavy chain single variable (V H ) domain antibodies are particularly preferred.
• Human heavy chain single variable V H are commonly abbreviated as V H domains.
• Single V H domains antibodies are also termed Humabody® herein.
• the isolated binding agents/molecules of the invention comprise or consist of at least one single domain antibody wherein said domain is preferably a human heavy chain variable domain.
• the binding agents of the invention comprise or consist of at least one human immunoglobulin single variable heavy chain domain; they are devoid of V L domains.
• Each single V H domain antibody comprises three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
• the domain is a human variable heavy chain (V H ) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
• the binding molecule includes an antigen binding fragment thereof.
• isolated single domain antibody refers to a single domain antibody that is substantially free of other single domain antibodies, antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.
• “Homology” generally refers to the percentage of amino acid residues in the candidate sequence that are identical with the residues of the polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percentage homology between two amino acid sequences is equivalent to the percentage identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known.
• antibody broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
• Ig immunoglobulin
• L light
• each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
• the heavy chain constant region is comprised of three domains, C H 1 , C H 2 and C H 3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM , IgD, IgA and IgY), class (e.g. , IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2) or subclass.
• type e.g., IgG, IgE, IgM , IgD, IgA and IgY
• class e.g. , IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2
• subclass e.g., IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2
• An antibody fragment is a portion of an antibody, for example as F(ab') 2 , Fab, Fv, sFv and the like. Functional fragments of a full length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs.
• scFv fragments ( ⁇ 25kDa) consist of the two variable domains, V H and V L . Naturally, V H and V L domain are non-covalently associated via hydrophobic interaction and tend to dissociate.
• stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).
• the smallest antigen binding fragment is the single variable fragment, namely the V H or V L domain. Binding to a light chain/heavy chain partner respectively is not required for target binding.
• Such fragments are used in single domain antibodies.
• a single domain antibody ( ⁇ 12 to 15 kDa) therefore has either the V H or V L domain.
• the isolated binding molecules of the invention comprise or consist of at least one single domain antibody wherein said domain is a V H domain.
• the binding molecules of the invention comprise or consist of at least one immunoglobulin single variable heavy chain domain antibody (sVD, sdAb or ISV) that has a V H domain, but is devoid of V L domains.
• the binding molecule may comprise two or more single V H domain antibodies.
• Such binding molecules may be monospecific or multispecific, monovalent or multivalent as explained in further detail below.
• the binding molecule does not comprise a light chain.
• the binding molecule does not comprise heavy chain domains C H 2 and C H 3.
• the binding molecule does not comprise a hinge region and heavy chain domains C H 2 and C H 3. In some embodiments, the binding molecule does not comprise heavy chain domains C H 1 , C H 2, and C H 3. In some embodiments the binding molecule does not comprise heavy chain domain C H 1 , a hinge region heavy chain domain C H 2 and heavy chain domain C H 3. In some embodiments the binding molecule does not comprise a light chain, a heavy chain domain C H 1 , a hinge region heavy chain domain C H 2 and heavy chain domain C H 3.
• Each V H domain comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• Modifications to the V H framework may be made to improve binding properties.
• the V H domain may comprise C or N-terminal extensions.
• the V H domain comprises C-terminal extensions of from 1 to 10, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids.
• the V H domain comprises C-terminal extensions of from 1 to 12 amino acid residues, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids of the C H 1 domain.
• said extension comprises at least 1 alanine residue, for example a single alanine residue, a pair of alanine residues or a triplet of alanine residues.
• V H domains are within the scope of the invention.
• V H domains that comprise additional C or N-terminal residues, for example linker residues and / or His tags, e.g., hexa- His (SEQ ID No. 251) or myc tags. Additional residues of the vector may also be present, for example in addition to tags. Binding molecules used may have the additional residues (SEQ ID No. 252).
• variable domain of the single domain antibodies of the invention is preferably a human variable domain (V H ).
• a human V H domain includes a fully human or substantially fully human V H domain.
• the term human V H domain also includes V H domains that are isolated from heavy chain only antibodies made by transgenic mice expressing fully human immunoglobulin heavy chain loci, in particular in response to an immunisation with an antigen of interest, for example as described in WO2016/062990 and in the examples.
• a human V H domain can also include a V H domain that is derived from or based on a human V H domain amino acid or nucleic acid sequence encoding such V H domain.
• variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences include variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences.
• a substantially human V H domain or V H domain that is derived from or based on a human V H domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo).
• the term "human V H domain” therefore also includes a substantially human V H domain wherein one or more amino acid residue has been modified.
• a substantially human V H domain the V H domain may include up to 10, for example 1 , 2, 3, 4 or 5 amino acid modifications compared to a fully human sequence.
• human V H domain or “substantially human V H domain”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• the term "human V H domain”, as used herein, is also not intended to include camelized V H domains, that is human V H domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the V H domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid V H H domain.
• variable domain refers to immunoglobulin variable domains defined by Kabat et a/. , Sequences of Immunological Interest, 5 th ed., U.S. Dept. Health & Human Services, Washington, D.C. (1991). The numbering and positioning of CDR amino acid residues within the variable domains is in accordance with the well-known Kabat numbering convention.
• the invention provides a single V H domain antibody or a binding molecule comprising one or more single V H domain antibody wherein said single V H domain antibody binds to human PSMA with an affinity, a Kon-rate, a Koff rate, KD and/or KA, EC50 and IC50 values as further described herein, in particular in the examples. Assays suitable for measuring these values are also shown in the examples.
• a binding molecule of the invention in particular the single V H domain antibody, comprises or consists of an amino acid sequence and preferred sequences and/or parts thereof, such as CDRs, as defined herein.
• CDR refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions.
• CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat is used herein.
• Kabat numbering Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e.
• single V H domain antibodies were isolated and grouped into families based on sequence homology in the CDR3 sequence. Through a process of optimization, a panel of variant single V H domain antibodies with a CDR sequence derived from a parent CDR sequence were also generated to improve affinities to PSMA and/or improve potencies compared to the parent molecule.
• the invention relates to a binding molecule capable of binding human PSMA comprising or consisting of or wherein the PSMA binding part comprises or consists of a single V H domain antibody comprising or consisting of a family 1 member as shown in table 1a or a family-1 like sequence, that is a variant of family 1 as shown in table 1a as defined herein.
• the binding molecule comprises or consists of at least one single V H domain antibody capable of binding PSMA, preferably human PSMA, comprising a family 1 single V H domain antibody as shown in table 1a or a variant of family 1 as shown in table 1a as defined herein.
• the invention relates to a single V H domain antibody capable of binding PSMA, preferably human PSMA, wherein said single V H domain antibody is as shown in table 1a or is a variant of family 1 as shown in table 1a as defined herein.
• the single V H domain antibody may include CDR1 , CDR2 and CDR3 sequences, as shown in below. CDR sequences and full length V H sequences in family 1 a are numbered according to
• NO. 1 NO. 2 3 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEVWSSIG
• NO. 5 NO. 6 7 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEVWSSIG
• NO. 9 NO. 10 11 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEWVSSIG TDYAADV ENDGTTDYAADVKGRFTISRDNSKNTL KG YLQMNSLRVEDTAVYYCVKDGVHWG
• NO. 13 NO. 14 15 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEWVSSIG TDYADVV ENDGTTDYADVVKGRFTISRDNSKNTL KG YLQMNSLRVEDTAVYYCVKDGVHWG
• NO. 17 NO. 18 19 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEWVSSIG TDYAAFV ENDGTTDYAAFVKGRFTISRDNSKNTL KG YLQMNSLRVEDTAVYYCVKDGVHWG
• NO. 21 NO. 22 23 EVQLLESGGGLVQPGGSLRLSCAASG SYALS SIGENDGT DGVH FSFSSYALSWVRQAPGKGLEVWSSIG
• NO. 25 NO. 26 27 EVQLLESGGGLVQPGGSLRLSCAASG
• NO. 33 NO. 34 35 EVQLLESGGGLVQPGGSLRLSCAASG
• Table 1a This shows sequences of single V H domain antibodies that are within the scope of the invention.
• the single V H domain antibody comprises CDR1 , 2, and 3 sequences as set out below.
• the single V H domain antibody may be selected from one of the following: a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 1 , a CDR2 sequence comprising SEQ ID NO. 2 and a CDR3 sequence comprising SEQ ID NO. 3; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 5, a CDR2 sequence comprising SEQ ID NO. 6 and a CDR3 sequence comprising SEQ ID NO.
• a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 9, a CDR2 sequence comprising SEQ ID NO. 10 and a CDR3 sequence comprising SEQ ID NO. 11 ; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 13, a CDR2 sequence comprising SEQ ID NO. 14 and a CDR3 sequence comprising SEQ ID NO. 15; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO. 17, a CDR2 sequence comprising SEQ ID NO. 18 and a CDR3 sequence comprising SEQ ID NO. 19; a single V H domain antibody comprising a CDR1 sequence comprising SEQ ID NO.
• the single V H domain antibody of the invention comprises a CDR1 sequence comprising SEQ ID NO. 25, a CDR2 sequence comprising SEQ ID NO. 26 and a CDR3 sequence comprising SEQ ID NO. 27.
• said single V H domain antibody comprises or consists of a sequence selected from SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40. In one embodiment, the single V H domain antibody comprises or consists of a sequence selected from SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40 or a sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto but wherein no changes are made to CDR2.
• said single V H domain antibody comprises a CDR2 as identified in any of SEQ ID NOs 2, 6, 10, 14, 18, 22, 26, 30, 34 or 38.
• said CDR2 is SEQ ID NO. 26.
• said modification(s) is in FR1 , CDR1 , FR2, CDR3, FR3, FR4.
• the single V H domain antibody has a V H domain that comprises or consists of SEQ ID NO. 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40.
• the binding molecules is selected from V H single domain antibodies 1.21 , 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29 or 1.30.
• the single V H domain antibody has a V H domain that comprises or consists of SEQ ID NO. 28.
• the invention also provides variant V H single domain antibodies that are variants of parent molecules 1.21 to 1.30 as shown in Table 1a, wherein these variants do not have a modification in the CDR2 sequence.
• Such variants have one or more amino acid substitution, deletion, insertion or other modification, and which retain a biological function of the parent single domain antibody.
• a variant V H single domain antibody retains binding to human PSMA.
• the parent single domain antibody can be sequence engineered to arrive at the variant. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence V H single domain antibody or polypeptide.
• Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
• Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
• a variant of a V H single domain antibody as used herein generally has at least 75%, 76%, 77%, 78%, 79%, 8 0%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the non-variant molecule, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
• Variants of a V H single domain antibody as shown in table 1a preferably do not have any modifications in the CDR2 region.
• the invention encompasses a V H single domain antibody as defined in SEQ ID NO. 41 or 48 and which has modifications at positions 62 and 63, but no modification at positions 55 and 56.
• Residues DS at positions 62 and 63 may for example be modified to AT, DF, DN, DA, AD, DV, AF, DT, DA, AS or AY.
• the modification in a variant is a conservative sequence modification.
• conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
• Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• one or more amino acid residues within the CDR regions of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (I) above) using the functional assays described herein.
• the invention therefore also comprises sequence optimised variants of the single domain antibodies described herein.
• modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence.
• a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived. To return one or more of the amino acid residues in the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis.
• Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.
• the glycosylation of an antibody is modified.
• an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
• Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
• Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
• one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
• Such aglycosylation may increase the affinity of the antibody for antigen.
• the family 1 or variants (family 1-like) binding molecules preferably have KD, Koff, KA, Kd, EC 50 and IC 50 values as further described herein and as shown in the examples.
• KD refers to the "equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon).
• KA refers to the affinity constant.
• the association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art.
• fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
• Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay and assays described in the examples can be used to test the binding molecules of the invention.
• the invention also relates to isolated nucleic acid sequences comprising or consisting of a sequence selected from SEQ ID NOs. 61 to 70 which encode V H domains 1.21 to 1.30.
• the sequence is SEQ ID NO. 67.
• Nucleic acid may include DNA and/or RNA.
• the present invention provides a nucleic acid that codes for a CDR or set of CDRs or a V H domain of the invention as defined above.
• a nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced.
• Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
• the invention relates to a nucleic acid construct comprising at least one nucleic acid defined above.
• the construct may be in the form of a plasmid, vector, transcription or expression cassette.
• the invention also relates to an isolated recombinant host cell comprising one or more nucleic acid construct as described above.
• the host cell may be a bacterial, yeast, viral, mammalian or other suitable host cell.
• the cell is an E. coli cell.
• the cell is a yeast cell.
• the cell is a Chinese Hamster Ovary (CHO) cell.
• a binding molecule described herein may be provided as a fusion protein with one or more additional protein moiety.
• the single domain antibody, described herein (a first moiety) may be provided as a fusion with a second moiety.
• the second moiety may comprise a V H domain that is also specific for human PSMA thus providing a bivalent binding molecule.
• the binding molecule is biparatopic.
• Biparatopic binding molecules comprise antigen-binding moieties that bind to different epitopes.
• Biparatopic binding molecules of the present invention can be constructed using methods known art. For example, to generate a bivalent binding molecule, two single domain antibodies of the invention may be connected, the two binding molecules may be from the same family or from different families of binding molecules of the invention. For example, a family 1 single V H domain antibody as described above and for example as shown in Table 1 or a variant thereof may be linked to a family 2 to 15 or family 2-like to 15- like single V H domain antibody.
• a family-like single V H domain antibody refers to a variant as defined elsewhere herein.
• a V H as defined for single V H domain antibody 1.27 (SEQ ID NO. 28) is connected to another single V H domain antibody selected from family 2 or family 3, for example to the single V H domain antibody as defined for clone 2.1.
• Two or more single V H domain antibody may be connected by a linker, for example a polypeptide linker.
• the invention relates to a binding molecule comprising a first single V H domain antibody capable of binding human PSMA with a CDR1 , 2 or 3 sequence as shown for any of V H 1.21 to 1.30 as shown in Table 1a or variant thereof (said variant preferably retaining one of the CDR2 sequences as set out in table 1 a) and a second single V H domain antibody capable of binding human PSMA.
• said single first domain V H domain antibody comprises a sequence selected from SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40.
• the second V H domain binds to the same epitope, part, domain, subunit or confirmation of PSMA as the first V H domain.
• the second V H domain may be selected from family 1 , 5, 6, 12 or 13 or family 1 , 5, 6, 12 or 13-like sequence.
• the first V H domain comprises or consists of SEQ ID No: 28.
• the second V H domain binds to a different epitope, part, domain, subunit or confirmation of PSMA than the first V H domain.
• the second V H domain may be selected from a family 2, 3, 4, 7, 9, 10, 11 or 14 or a family 2, 3, 4, 7, 9, 10, 11 or 14-like sequence.
• said second V H domain antibody is selected from Table 2.
• the first V H domain comprises SEQ ID No:28 and the second V H domain comprises SEQ ID NO:71.
• said second V H domain antibody is selected from Table 2.
• the second moiety may comprise a V H domain or another antibody fragment that is specific for a different antigen to provide a bispecific binding molecule.
• the term "bispecific binding molecule” thus refers to a polypeptide that comprises a binding molecule as described herein which has a binding site that has binding specificity for PSMA, and a second polypeptide domain which has a binding site that has binding specificity for a second target, i.e., the bispecific binding molecule has specificity for two targets.
• the first target and the second target are not the same, i.e. are different targets, e.g., proteins; both may be present on a cell surface.
• a bispecific binding molecule as described herein can selectively and specifically bind to a cell that expresses (or displays on its cell surface) the first target and the second target.
• the binding molecule comprises more than two antigen-binding moieties.
• first and second do not designate the orientation of the molecule, that is the first VH may be C or N-terminally located.
• more than two moieties are joined together providing a multispecific binding molecule.
• a multispecific polypeptide agent as described herein can in addition to binding PSMA bind one or more additional targets, i.e., a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target binding sites respectively.
• the term "target” refers to a biological molecule (e.g., antigen, peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain which has a binding site can selectively bind.
• the target can be, for example, an intracellular target (such as an intracellular protein target) or a cell-surface target (such as a membrane protein, e.g., a receptor protein).
• a target is a cell-surface target, such as a cell-surface protein.
• the first cell-surface target and second cell-surface target are both present on a cell.
• the target is an immunooncology target.
• Multispecific antibodies of the present invention can be constructed using methods known art. If desired, bispecific or multispecific binding molecules can be linked to an antibody Fc region or fragment thereof, comprising one or both of C H 2 and C H 3 domains, and optionally a hinge region. For example, vectors encoding bispecific or multispecific binding molecules linked as a single nucleotide sequence to an Fc region or fragment thereof can be used to prepare such polypeptides.
• the second moiety may serve to prolong the half-life of the binding molecule.
• the second moiety may comprise a protein, for example and antibody, or part thereof that binds a serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA).
• the second moiety may comprise a V H domain that binds serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA).
• the second moiety may comprise a serum albumin, e.g. a human serum albumin (HSA) or a variant thereof such as HSA C34S.
• binding molecule as described herein comprising a V H domain and an Fc domain, e.g., wherein the V H domain is fused to an Fc domain.
• a binding molecule that comprises a second variable domain that specifically binds a second antigen, where the second antigen is an antigen other than human PSMA.
• the second antigen may be a cluster of differentiation (CD) molecule or a Major Histocompatibility Complex (MHC) Class II molecule.
• the binding molecule of the invention is labelled with a detectable or functional label.
• a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.
• the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
• the binding molecule of the invention is coupled to at least one therapeutic moiety, such as a drug, an enzyme or a toxin.
• the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin.
• the PSMA binding molecule of the invention can be coupled to a radioactive isotope such as an ⁇ -, ⁇ -, or ⁇ -emitter, or a ⁇ - and ⁇ -emitter.
• the toxin as used in the various aspects and embodiments of the invention may be selected from calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA- C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin PHE.
• the therapeutic moiety is an immunostimulatory or immunomodulating agent.
• the invention thus provides an immunoconjugate comprising a single V H domain antibody described herein.
• the invention relates to an immunoconjugate of the formula A-(L-D)n wherein A is an antigen-binding moiety comprising a first human single heavy chain variable immunoglobulin (V H ) domain antibody capable of binding specifically to human PSMA as described herein, optionally comprising a second human single heavy chain variable immunoglobulin (V H ) domain antibody capable of binding specifically to human PSMA and optionally comprising a third human single heavy chain variable immunoglobulin (V H ) domain antibody, L is a linker, and D is an auristatin or a derivative thereof and n is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
• the single V H domain antibody comprising a CDR1 , CDR2 and CDR3 sequence as described herein can be used in an immunoconjugate as described above.
• Said single domain V H domain antibody comprises a sequence selected from SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36 or 40.
• the sequence is SEQ ID No. 28 and the V H is V H 1.27.
• said second single domain V H domain antibody is selected from Family 2.
• D is MMAE, MMAF, or a derivative thereof.
• D is MMAE or a derivative thereof conjugated to the antigen-binding moiety via a valine-citrulline (vc) linker (vc-MMAE).
• D is MMAF or a derivative thereof conjugated to the antigen-binding moiety via a maleimidocaproyl linker (mc-MMAF).
• L- D is vedotin or mafodotin.
• the immunoconjugates of the invention are preferably of the formula A-(L-D)n wherein A is an Auristatin or derivative thereof.
• D is MMAE, MMAF, or a derivative thereof.
• Auristatins are synthetic analogues of the antineoplastic natural product Dolastatin. Auristatins inhibit cell division by blocking the polymerisation of tubulin and are used as toxic payloads in antibody-drug conjugates.
• the family of auristatins includes monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). In preclinical models, auristatins have been found to be 100- to 1 ,000-fold more potent than traditionally-used chemotherapeutics.
• MMAE Monomethyl auristatin E
• MMAE desmethyl-auristatin E
• the lUPAC name for MMAE is (S)-N-((3R,4S,5S)-1-((S)-2-((1 R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin- 1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamido)butanamide.
• Monomethyl auristatin E or MMAE is 100-1000 times more potent than doxorubicin, but its toxicity is such that cannot be used as a drug itself. However, it has been used as part of an antibody-drug conjugate or ADC, wherein MMAE is linked to a monoclonal antibody (mAb) that recognizes a specific marker expressed in cancer cells and directs MMAE to the cancer cell.
• mAb monoclonal antibody
• MMAE As MMAE is toxic, it has been used as a therapeutic only when conjugated to a monoclonal antibody (mAb) to target the MMAE to cancer cells.
• mAb monoclonal antibody
• the name "vedotin” denotes MMAE plus its linking structure to the antibody.
• the structure linking the targeting mAb to MMAE may comprise an attachment group (maleimide (mal) and caproic acid (cap)), a spacer (paraaminobenzoic acid) and a cathepsin-cleavable linker (amino acids valine (Val) and citrulline (Cit)).
• the tether that connects MMAE to the monoclonal antibody is stable in extracellular fluid, but is cleaved by cathepsin once the antibody-drug-conjugate has bound to the targeted cancer cell antigen and entered the cancer cell, after which the ADC releases the toxic MMAE and activates the potent anti-mitotic mechanism.
• Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of highly potent cytotoxic agents.
• Monomethyl auristatin F (MMAF, desmethyl-auristatin F) is a synthetic antineoplastic agent.
• the lUPAC name for MMAF is (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2- ((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5- methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid.
• MMAF is the toxic payload used in some experimental anti-cancer antibody-drug conjugates such as vorsetuzumab mafodotin and SGN-CD19A.
• mafodotin refers to MMAF plus its attachment structure to the antibody.
• the attachment group may consist of maleimide and caproic acid.
• Auristatins and their use as components of ADC are reviewed by Maderna and Leverett in “Recent Advances in the Development of New Auristatins: Structural Modifications and Application in Antibody Drug Conjugates”; Mol. Pharmaceutics, 2015, 12 (6), pp 1798-1812 Mendelsohn et al., "Investigation of Hydrophilic Auristatin Derivatives for Use in Antibody Drug Conjugates".
• Bioconjugate Chem., Article ASAP DOI: 10.1021/acs.bioconjchem.6b00530, Publication Date (Web): January 6, 2017 describe derivatives of the natural product dolastatin 10 containing pyridines and other basic amines, which were examined to assess more hydrophilic auristatin derivatives would be sufficiently potent for use in ADC.
• a pyridine derivative, monomethyl auristatin PYE showed the greatest potency when tested in vivo.
• the immunoconjugate, compositions and methods of the invention may feature an auristatin which is either monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) or a derivative thereof.
• MMAE monomethylauristatin E
• MMAF monomethylauristatin F
• MMAE may be conjugated to the antigen-binding moiety via a valine-citrulline (vc) linker (vc- MMAE).
• MMAF is conjugated to the antigen-binding moiety via a maleimidocaproyl linker (mc-MMAF) using HiPEGTM technology (WO 2009/047500; Cong et al. , (2012) Bioconjugate Chem. 2012, 23, 248-263.
• D is MMAE or a derivative thereof conjugated to the antigen- binding moiety via a valine-citrulline (vc) linker (vc-MMAE).
• D is MMAF or a derivative thereof conjugated to the antigen-binding moiety via a maleimidocaproyl linker (mc-MMAF).
• L-D is vedotin or mafodotin. L- D may also comprise one of more H amino acid which links A and L-D.
• the immunoconjugate may comprise a further toxic moiety, a label, half life extension or other moiety.
• the immunoconjugate may be used in the manufacture of the manufacture of a medicament for the treatment of a cancer associated with expression of PSMA, prostate cancer or a prostatic disorder.
• the invention also relates toimmunoconjugate as described above for treatment of a disease as described herein.
• Toxin-conjugated forms of the PSMA binding molecules of the present invention preferably mediate specific cell killing of PSMA-expressing cells at picomolar concentrations.
• the PSMA binding molecules of the invention are modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome or using a serum albumin protein.
• the binding molecule of the invention is covalently modified.
• covalently modified/covalent modification includes modifications of a binding molecule according to the present invention, e.g., of a specified sequence herein; with an organic proteinaceous or non-proteinaceous derivatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications.
• Covalent modified polypeptides still have the functional properties described herein, for example the ability to bind the human PSMA or
• Covalent modifications are generally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells.
• Certain post- translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post- translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deaminated under mildly acidic conditions.
• post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues, methylation of the [alpha]-amino groups of lysine, arginine, and histidine side chains.
• Covalent modifications include fusion proteins comprising a PSMA binding molecule according to the present invention, e.g., of a specified sequence and their amino acid sequence variants, such as immunoadhesins, and N-terminal fusions to heterologous signal sequences.
• binding molecules of the invention have certain functional properties as further described below. These and other pharmacological activities of the binding molecules of the invention may be demonstrated in standard test methods for example as described in the art.
• binding molecules of the invention can be internalised into a cell along with the prostate- specific membrane antigen. Binding molecules of the invention bind specifically to epitopes on the extracellular domain of human PSMA. In one embodiment, binding molecules of the invention specifically bind PSMA in its dimeric form. Binding molecules of the invention can be conjugated to a toxic moiety and used to ablate or kill PSMA-expressing prostatic or cancerous cells. Binding molecules of the invention can bind live cells, such as a tumor cell or a prostate cell, such as human PSMA expressing CHO cells, LNCaP cells as shown in the examples (see examples 7b and Tables 18 and 19).
• the present invention provides single domain antibodies that bind to PSMA with an EC50 value of between 100 nM and 100 pM, such as at an average EC50 value of 100nM or less, even more preferably at an average EC50 value of 90 nM or less, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, or 1 nM or even less, such as less than 500, 400, 300, 200, 100 pM, or even less, such as less than 4 pM, preferably as measured in a FMAT binding assay.
• EC50 values are shown in Table 19.
• binding molecules of the invention are capable of binding specifically to human PSMA and to cynomolgus monkey PSMA.
• IC 50 is the concentration of a binding member that reduces a biological response by 50% of its maximum.
• IC 50 may be calculated by plotting % of maximal biological response as a function of the log of the binding member concentration, and using a software program to fit a sigmoidal function to the data to generate IC 50 values. Methods for measuring IC 50 are well known in the art. For example, to determine the IC 50 , a HIS ZAP Cell Killing assay may be employed to determine IC 50 . EC 50 designates the half maximal effective concentration.
• the invention relates to a binding molecule comprising or consisting of at least one immunoglobulin single domain antibody directed against PSMA, preferably human PSMA, wherein said domain is a human V H domain and has an IC 50 of about 0.2 to about 1000 nM or more, for example 0.2 to 900, 0.2 to 800, 0.2 to 700, 0.2 to 600, 0.2 to 500, 0.2 to 400, 0.2 to 300, 0.2 to 200, 0.2 to 100, 0.2 to 50, 0.2 to 40, 0.2 to 30, 0.2 to 20, 0.2 to 10, 0.2 to 9, 0.2 to 8, 0.2 to 7, 0.2 to 6, 0.2 to 5, 0.2 to 4, 0.2 to 3, 0.2 to 2 or 0.2 to 1 when tested as described in the examples.
• binding kinetics and affinity (expressed as the equilibrium dissociation constant, KD) of PSMA binding molecules of the invention for binding PSMA may be determined, e.g., using surface plasmon resonance such as BIAcore® or Octet, or KD may be estimated from pA2 analysis.
• the molecules of the invention are very potent (i.e., EC50 values as measured, e.g., in the experimental part in the pM range).
• the present invention provides a single domain antibody as described herein, wherein said sdAb binds to said PSMA with an average KD value of between 100 nM and 10 pM, such as at an average KD value of 90 nM or less, even more preferably at an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM, or even less such, as less than 10 pM.
• the KD is determined as shown in the examples (example 8b).
• a binding molecule according to the invention has a binding affinity to PSMA with an affinity constant of at least about 10 7 M "1 , preferably about 10 9 M '1 , and more preferably, about 10 10 M "1 to 10 11 M “1 or higher.
• a binding molecule according to the invention has a Kon of 1.00E+04 to 1.00E+6 (1/Ms).
• a binding molecule according to the invention has Koff of 1.00E-03 to 1.00E-05 (1/s).
• Binding molecules of the invention have shown excellent stability, including heat and serum stability (see examples). Furthermore, binding molecules of the invention show rapid tumor targeting as shown in the examples. Furthermore, binding molecules of the invention also show high specificity for human PSMA and low uptake in non-target tissues (see examples).
• binding molecules of the invention show fast blood clearance. In one embodiment, binding molecules of the invention show low renal retention. In one embodiment, binding molecules can inhibit, e.g., competitively inhibit, the binding of another antibody e.g., J591 , to human PSMA.
• another antibody e.g., J591
• the binding molecule of the invention as defined herein may have one or more property select from the following non-limiting list:
• Methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries can comprise the steps of: a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; and
• the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
• suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art (see for example Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1 st edition (October 28, 1996) Brian K. Kay, Jill Winter, John McCafferty).
• a binding molecule described herein, including heavy chain antibody with a V H domain can be expressed in a transgenic rodent, for example a mouse.
• the transgenic rodent for example a mouse, may have a reduced capacity to express endogenous antibody genes.
• the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes.
• the rodent may therefore comprise modifications to disrupt expression of endogenous light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced.
• a pharmaceutical composition comprising a PSMA binding molecule according to the present invention and optionally a pharmaceutically acceptable carrier.
• the binding molecule of the present invention or compositions can be administered by any convenient route.
• the compounds may be administered by any route, including oral and parenteral administration.
• Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration.
• Compositions can take the form of one or more dosage units.
• the composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension.
• the liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.
• the liquid compositions of the invention can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin
• a composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.
• it can be desirable to administer one or more binding molecule of the present invention or compositions locally to the area in need of treatment, or by intravenous injection or infusion.
• the amount of the binding molecule of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
• compositions of the invention comprise an effective amount of a binding molecule of the present invention such that a suitable dosage will be obtained.
• the correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.
• this amount is at least about 0.01 % of a binding molecule of the present invention by weight of the composition.
• Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present invention.
• the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.
• compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use.
• suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
• the pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a binding molecule of the present invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.
• the invention furthermore relates to a method for the prevention and/or treatment of cancer, in particular prostate cancer, comprising administering a binding molecule of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a binding molecule and/or of a pharmaceutical composition of the invention.
• the invention relates to a method for the prevention and/or treatment of cancer, in particular prostate cancer, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a binding molecule or a pharmaceutical composition of the invention.
• the invention also relates to a binding molecule of the invention for use in the treatment of disease.
• the invention also relates to a binding molecule of the invention for use in the treatment of cancer, in particular prostate cancer or a prostatic disorder.
• Prostate cancer refers to all stages and all forms of cancer arising from the tissue of the prostate gland.
• the invention also relates to the treatment of a disease characterized by aberrant expression of PSMA.
• the invention relates to the use of a binding molecule of the invention in the treatment of disease. In another aspect, the invention relates to the use of a binding molecule of the invention in the manufacture of a medicament for the treatment of cancer, in particular prostate cancer or a prostatic disorder.
• the binding molecules of the invention are also useful for the treatment, prevention, or amelioration of cancer, in particular prostate cancer or a prostatic disorder.
• a prostatic disorder refers to any disease that afflicts the prostate gland in the male reproductive system. The prostate gland is dependent on the hormonal secretions of the testes. Expression of PSMA has been detected in other cancers, more specifically in the neovasculature associated with these cancers.
• carcinomas including conventional (clear cell) renal cell, transitional cell of the bladder, testicular-embryonal, neuroendocrine, colon, and breast, and the different types of malignancies were found consistently and strongly to express PSMA in their neovasculature.
• the binding molecule of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic and/or cytotoxic moiety.
• the binding molecule may be conjugated to a toxic moiety.
• the anti-PSMA binding molecule can be used in combination with existing therapies.
• the single domain antibody is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy.
• the invention also relates to a combination therapy comprising administration of a single domain antibody or pharmaceutical composition of the invention and an anti-cancer therapy.
• the anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs.
• therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g.
• modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens including EGFR antagonists
• an anti-inflammatory agent including a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy.
• the single domain antibody is used in combination with surgery.
• the binding molecule of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.
• the invention provides a kit for detecting prostate cancer for diagnosis, treatment, prognosis or monitoring comprising a binding molecule of the invention.
• the kit may also comprise instructions for use.
• the kits may include a labeled binding molecule of the invention as described above and one or more compounds for detecting the label.
• the invention in another aspect provides a binding molecule of the invention packaged in lyophilized form, or packaged in an aqueous medium.
• the invention also relates to detection methods using the binding molecule of the invention.
• the human-PSMA-binding molecules, disclosed herein can be used to detect PSMA (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry.
• ELISA enzyme linked immunosorbent assays
• RIA radioimmunoassay
• tissue immunohistochemistry tissue immunohistochemistry
• the invention also relates to in vitro or in vivo methods for diagnosing or monitoring progression of a cancer, in particular prostate cancer.
• In vitro methods comprise detecting the presence of a PSMA protein in a test sample and comparing this with control sample from a normal subject or with a standard value or standard value range for a normal subject.
• the sample may be selected from blood, plasma, serum, semen, urine or a tissue biopsy.
• the method may include: (a) contacting the sample (and optionally, a reference, e.g., a positive and/ or negative control sample) with a PSMA binding molecule of the invention and (b) detecting either the binding molecule bound to PSMA or unbound binding molecule in the sample, to thereby detect PSMA in the biological sample.
• the binding molecule can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
• In vivo methods may comprise detecting the presence of PSMA in vivo, for example by imaging in a subject.
• a PSMA binding molecule of the invention is labeled to detect binding.
• human PSMA can be assayed in biological fluids by a competition immunoassay utilizing PSMA standards labeled with a detectable substance and an unlabeled human PSMA binding molecule.
• the biological sample, the labeled PSMA standards and the human PSMA binding molecule are combined and the amount of labeled PSMA standard bound to the unlabeled binding molecule is determined.
• the amount of human PSMA in the biological sample is inversely proportional to the amount of labeled PSMA standard bound to the PSMA binding molecule.
• human PSMA can also be assayed in biological fluids by a competition immunoassay utilizing PSMA standards labeled with a detectable substance and an unlabeled human PSMA binding molecule.
• Binding molecules disclosed herein can be used to inhibit PSMA activity, e.g., in a cell culture containing PSMA, in human subjects or in other mammalian subjects having PSMA with which a binding molecule disclosed herein cross-reacts.
• a method for inhibiting or increasing PSMA activity comprising contacting PSMA with a binding molecule disclosed herein such that PSMA activity is inhibited or increased.
• a binding molecule disclosed herein can be added to the culture medium to inhibit PSMA activity in the culture.
• the invention also relates to a method of ablating or killing a cell that expresses PSMA, e.g., a cancerous or non-cancerous prostatic cell.
• Methods of the invention include contacting the cell, with PSMA binding molecule of the invention, in an amount sufficient to ablate or kill, the cell.
• the methods can be used on cells in culture, e.g., in vitro or ex vivo.
• mice carrying a heavy-chain antibody transgenic locus in germline configuration within a background that is silenced for endogenous heavy and light chain antibody expression were created as previously described (WO2004/076618 and WO2003/000737, Ren ef a/., Genomics, 84, 686, 2004; Zou et al., J. Immunol., 170, 1354, 2003, WO2016/062990).
• transgenic mice were derived following pronuclear microinjection of freshly fertilised oocytes with a yeast artificial chromosome (YAC) comprising a plethora of human V H , D and J genes in combination with mouse immunoglobulin constant region genes lacking C H 1 domains, mouse enhancer and regulatory regions.
• YAC yeast artificial chromosome
• the immunisations used recombinant purified protein or Human Cell Line LNCap.
• Recombinant human PMSA was purchased from R&D, (cat. no. 4234-ZN), while the LNCap cells were from Sigma Aldrich (cat. no. 89110211-1 VL).
• Tg/TKO mice aged 8 - 12 weeks of age each received a total of 50 ⁇ g of recombinant purified human PSMA protein, emulsified in Complete Freund's Adjuvant and delivered subcutaneously, or 10 million LNCap cells in PBS delivered intraperitoneal ⁇ , followed by boosts of 1 - 10 ⁇ g of the recombinant protein, emulsified in Incomplete Freund's Adjuvant, also administered subcutaneously, given at various intervals following the initial priming.
• a final dose of the recombinant purified human PSMA protein antigen was administered intraperitoneally, in phosphate buffered saline, in the absence of adjuvant.
• Alternative immunisation routes and procedures can also be employed.
• DNA immunisations are often delivered intramuscularly or via a Genegun.
• Transfected cells or membrane preparations from such cells are often, although not exclusively, administered intraperitoneally.
• RNAIate® Spleen, inguinal and brachial lymph nodes were collected into RNAIate® from each immunised animal. For each animal, 1/2 of the spleen and 4 lymph nodes were processed separately. Initially, the tissues were homogenised; following transfer of tissues to Lysing matrix D bead tubes (MP Bio. Cat. no. 1 16983001), 600 ⁇ of RLT buffer containing ⁇ - mercaptoethanol (from Qiagen RNeas® kit cat. no. 74104) was added before homogenisation in a MP Bio Fastprep96 homogeniser (cat# 116010500) at 1600rpm for 60 seconds. The tubes containing the homogenised tissues were transferred to ice and debris was pelleted by centrifugation at 1200rpm for 5 minutes.
• Lysing matrix D bead tubes MP Bio. Cat. no. 1 16983001
• 600 ⁇ of RLT buffer containing ⁇ - mercaptoethanol from Qiagen RNeas® kit cat. no.
• RNA sample was extracted using Qiagen RNeasy® kit (cat. no. 74104) following the manufacturer's protocol. Each RNA sample was then used to make cDNA using Superscript II I RT-PCR high-fidelity kit (Invitrogen cat. no. 12574-035). For each spleen and lymphnodes RNA sample, 5 RT-PCR reactions were performed, each with VH_J/F (long) primer in combination with a primer for V H 1 , V H 2, V H 3, V H 4 or V H 6 family. Details of the primers are below. Table 16. Primers for V10:
• Residues in bold have homology with pUCG3
• Residues in bold have homology with pUCG3
• R A, G. Y: C, T. M: A, C. K: G, T. S: C, G. W: A, T. B: C, G, T. V : A, C, G. D : A, G, T. N: A, C, G, T, T
• the phagemid vector, pUCG3, was employed in these studies.
• a PCR-based method was used to construct the V H phagemid libraries from the amplified V H sequences. The following procedure was used: A linearised version of pUCG3 was created using PCR; with the following primers: pUCG3-pH ENAPmut4 SEQ ID No. 247 pUCG3- pHENAPmut5mycHis SEQ I D No. 248
• Phusion High fidelity PCR master mix with GC buffer (cat. no. F532L, NEB) was used for the PCR reactions.
• the PCR product (3152bp) was gel purified using Fermentas GeneJet Gel purification kit (cat. no. K0691), according to the manufacturer's instructions, with final elution in 40 ⁇ of elution buffer.
• the purified V H RT-PCR products were employed as megaprimers with the linearised pUCG3 to give phagemid products for transformation and library creation.
• Phage and cells were incubated on ice for 2 hours, mixing occasionally to prevent cell clumping. Unbound or weakly bound phage were removed by washing five times in ice-cold PBS. The phage were then allowed to internalise by incubating the cells in media at 37°C before removing phage bound to the outside of the cells with a 5 minutes wash step in a low pH cell-stripping buffer at 4°C. The cells were then lysed to harvest internalised phage using trimethylamine. Both the stripped and internalised fractions were neutralised with Tris buffer before being used to infect E.coli. The phage outputs were analysed as described for panning selections on recombinant proteins. EXAMPLE 7. Assays for target binding
• V H from the different selections were screened in one or more of the following assays to identify specific V H capable of binding PMSA.
• V H antibodies were identified by phage ELISA following published methods (Antibody Engineering, edited by Benny Lo, chapter 8, p161 - 176, 2004). Phage ELISAs were performed against target protein and an unrelated antigen as control. In some cases, purified or crude extracts of V H domains were assayed by ELISA instead of using a phage ELISA. In these cases, bacterial periplasmic extracts or purified V H were used. b) FMAT Direct cell Binding Assay
• Periplasmic extracts from E.coli were screened for production of PSMA-binding-His-tagged
• V H Peri preps were tested by single point screening for the presence of V H that bound specifically to CHO human PSMA, CHO cyno PSMA and LnCAP cells with no binding to CHO parental cells in an FMAT Direct Binding Assay.
• V H purified via the terminal His tag were serially diluted in FMAT assay buffer then binding was measured as described above ( Figure 1).
• Improved variants show similar properties to the parent V H ( Figure 1 b and 1 c).
• Table 18 EC50 values for anti-PSMA V H binding to PSMA expressing cell lines. Values are in the picomolar range (prepared from purified V H )
• V H clone as identified above was sequenced from the phagemid and grouped based on V H germline and CDR3 amino acid similarity into separate families. Representative clones were further characterised. Variants, including germlined variants, were generated by standard methods of parent clones e.g. 1 .1 or 2.1 . Table 1 shows the sequences of clones family in family 1. Clones 1.8-1.30 are variants of 1 .1 . Clones 1.21 to 1.30 are improved sequence optimised variants with liability corrections in the CDR2 sequence.
• V H were tested for binding to PMSA and shown not to cross react with irrelevant proteins.
• RED 384 instrument Recombinant PMSA was diluted to 20pg/ml in sodium acetate buffer, pH 5 (ForteBio, cat. no. 18-1069) and coupled to ARG2G biosensors (ForteBio cat. no. 18- 5092) using amine-coupling chemistry (NHS-EDC amine-coupling, ForteBio cat. nos. 18- 1067 and 18-1033), followed by quenching in ethanolamine (ForteBio cat. no. 18-1071).
• amine-coupling chemistry NHS-EDC amine-coupling, ForteBio cat. nos. 18- 1067 and 18-1033
• Binding kinetics of anti-PSMA V H antibodies were then determined by preparing each V H antibody in dilution series (typically 1 :2 dilution series starting with 15pg/ml, V H at the highest concentration), and then measuring binding of the different V H concentrations to the PSMA- coupled biosensors. V H binding kinetics were then determined from the (blank subtracted) sensorgram trace using 1 :1 binding models and ForteBio Octet DataAnalysis software. Binding affinities from 1-150nM and in the subnanomolar range were detected and examples of the Octet profiles are shown in Figure 2 and in the binding parameters thereof in Table 19 below.
• V H Internalization of purified V H was measured using the pH-sensitive fluorescent dye pHrodo® green. Anti-His antibody (Millipore cat. no. 05-949) was labelled with pHrodo® Green STP ester (Molecular Probes cat. no. P35369) according to the manufacturer's instructions. All samples and reagents were prepared in internalization buffer (pH 7.4) containing PBS and 0.1 % Bovine Serum Albumin. CHO cells expressing cynomolgus PSMA were resuspended at 0.1 x 10 s cells/ml and 120 nM DRAQ5 added to the cell suspension. V H (10 ⁇ ) were transferred into 384-well black clear-bottomed assay plates (Costar cat. no.
• CHO cells expressing human or cynomolgus PSMA 400 cells per well in a 30 ⁇ volume were seeded into 384-well black clear-bottomed tissue culture-treated assay plates (Costar cat. no. 3712) in Hams F12 (Sigma cat. no. N6658) media containing 10% foetal bovine serum, 2mM L-glutamine, 10pg/ml blasticidin, 300pg/ml Zeocin, penicillin/streptomycin, 1 pg/ml tetracycline and incubated overnight in a C0 2 incubator at 37°C. Purified V H were serially diluted in media then an equal volume of 40nM His-ZAP added.
• V H /His-ZAP samples (10 ⁇ ) were transferred to the cell assay plates and incubated for either 72 or 48 hours in a C0 2 incubator at 37°C.
• His-ZAP control wells cells with His-ZAP reagent
• background controls media only
• Cell viability was determined following either 72 or 48 hour incubation using the Cell Titer-Glo Cell Viability assay (Promega cat. no. G7571) according to the manufacturer's instructions.
• Relative luminescent signal (RLU) was measured using the BMG PHERAstar plate reader. The data was normalized by subtraction of the RLU signal obtained in the absence of cells and expression as a percentage of the background- corrected signal of the His-ZAP control wells. Examples are given in Figure 4A&B.
• V H from the different CDR3 families were tested for developability characteristics.
• V H Serum stability was assessed using a Homogenous Time Resolved
• V H Purified V H were mixed with cynomolgus monkey serum and incubated for 0-7 days at 37°C.
• DSC Differential scanning calorimetry
• V H were injected in mice (V H 1.1 , V H 2.1 and V H 2.1 with half-life extension).
• the mice contain PSMA positive (+) and PSMA negative (-) tumours. Studies were carried out as follows:
• the half-life extended V H comprises an anti-mouse serum albumin (anti-MSA) V H with the following sequence: SEQ ID NO: 249.
• anti-MSA anti-mouse serum albumin
• All V H domains used in this study were expressed in E.coli.
• the proteins were purified from filtered supernatant using nickel affinity chromatography and size exclusion chromatography (SEC) as described in example 7a. After buffer exchange into storage buffer, the some proteins were concentrated using spin concentrators. The protein purity was analysed using SDS-PAGE and analytical SEC. Binding to PSMA was checked using recombinant protein and/or cells expressing PSMA. Stability was checked by heating the protein to 40°C for an extended period of time (ranging from overnight to 4 weeks) and measuring the degree of protein degradation. Aliquots of the proteins were stored at -80°C until use.
• the cell line used was a CHO T-REx huPSMA cell line.
• Lysosomes and endosomes are stained using a primary antibody against either the early endosome antigen 1 (EEA-1) or the lysosome membrane antigen 1 (LAMP-1) (both rabbit) for 1 hr. Cells are further incubated for 1 hr RT with anti-rabbit-647 secondary antibody, followed by washes.
• EAA-1 early endosome antigen 1
• LAMP-1 lysosome membrane antigen 1
• Coverslips are mounted into frosty end slides and imaged using a NI KON A1 R confocal system.
• Laser lines used were: 407.7nm (HOECHST), 487.7nm (VH/monoclonal benchmark), 639.7 (LAMP-1 , EEA-1 )
• VH VH were added to the plates and incubated at 4°C for 30 minutes following by 37°C for 2 hours. Plates were washed three times with PBS then the cells fixed in 4% paraformaldehyde and permeabilised with 0.5% saponin. Internalized VH were detected by staining with anti-His (Millipore 05-949) and anti-mouse AF488 (Jackson ImmunoResearch 1 15-545-098). Lysosomes were stained with LAMP-1 (Abeam Ab24170) and anti-rabbit AF647 (Jackson ImmunoResearch 11 1 -605- 008). Nuclei were stained using Hoescht stain (Life technologies H3570). Plates were imaged using the I N Cell Analyzer 6000 and Images processed using ImageJ software.
• MMAE-toxin-conjugated V H was determined using an in vitro cytotoxicity assay.
• Human cells DU-145, ATCC HTB-81 ) stably expressing human PSMA or matched PSMA negative cells were seeded into 384-well black clear-bottomed tissue culture treated assay plates at 3000 cells per well in RPMI 1640 medium containing 10% foetal bovine serum, 2mM L-Glutamine, 1X penicillin/streptomycin, and incubated overnight in a C0 2 incubator at 37°C. Cells were then incubated with serially-diluted M MAE-toxi n-conj ugated V H for 48 or 72 hours.
• Untreated control wells (cells in the absence of toxin-conjugated V H ) and background control wells (media only) were set up on each plate for data normalization.
• Cell killing was determined following the incubation using the Cell Titer-Glo Cell Viability assay (Promega G7571) according to the manufacturer's instructions.
• Relative luminescent signal (RLU) was measured using the BMG PHERAstar plate reader. The data was normalized by subtraction of the RLU signal obtained in the background control wells then expressed as a % of the untreated control wells (% survival).
• Figure 16 illustrates dose response curves obtained using a human-PSMA-expressing human cell line and the matched parent (i.e.
• the anti-PSMA-MMAE-conjugated V H specifically killed PSMA positive cells with minimal cell killing observed for the PSMA negative control cell line.
• the biparatopics that consist of two V H targeting different epitopes of the PSMA were more potent than the monovalent or bivalent PSMA V H constructs.
• Table 31 Summary of in vitro cytotoxicity data obtained with the human-PSMA-expressing human cell line following a 48 hour incubation.
• Hi PEGTM D-His 6 val-cit- Monovalent HEL4-bis >300nM (n 3)
• HiPEGTM B-2-B-His 6 val- Bivalent 1.1- 0.7 18 ⁇ 8nM 12.6 cit-PAB-MMAE (G4S)6- (n 3)
• Table 32 Summary of in vitro cytotoxicity data obtained with the human-PSMA-expressing human cell line following a 72 hour incubation.
• HDCs HumabodyTM drug conjugates
• a stock solution of conjugation reagent, HiPEGTM val-cit-PAB-MMAE (Figure 18), was prepared in MeCN prior to performing conjugation reactions.
• a solution of HumabodyTM (0.9 mg/mL in PBS; 20 mM EDTA, pH 7.5) was mixed gently with HiPEGTM val-cit-PAB-MMAE reagent (1.5 equiv. per HumabodyTM; 5% (v/v) final MeCN concentration) and incubated at 22 °C for 19 h. After 19 h, the conjugation reaction was mixed with an equal volume of 600 mM sodium phosphate buffer (150 mM NaCI; 20 mM EDTA), pH 7.5 and cooled to 4 °C.
• a stock solution of 1 mg/mL NaBH 4 solution was prepared in 0.1 M NaOH. Two aliquots each of NaBH 4 solution, (10 equiv. per reagent), were added to the cooled conjugation reaction with a 30 min interval between additions. After a further 30 min interval, the crude mixture was purified by hydrophobic interaction chromatography (HIC) using a TOSOH ToyoPearl Phenyl-650S column. The sample was bound and washed onto the column using 50 mM sodium phosphate (2 M NaCI), pH 7 (buffer A) and eluted using a gradient of 50 mM sodium phosphate (20% v/v isopropanol), pH 7 (buffer B).
• HIC hydrophobic interaction chromatography
• Fractions containing the mono-loaded product were pooled and concentrated using Vivaspin20 concentrators fitted with 5 kDa MWCO PES membranes.
• the concentrated fractions were buffer exchanged into DPBS using PD10 columns and the buffer exchanged material sterile filtered using 0.2 ⁇ PVDF syringe filtration unit.
• the HiPEG val-cit-PAB-MMAE moiety is attached via a C terminal His6-tag on a V H .
• Two histidines are needed for attachment of each "payload" toxin molecule.
• Positive control antibody Pro_006 is an anti-PSMA antibody composed of heavy and light chain sequences described within US8470330 and exemplified as antibody 006.
• Conjugation 1 A solution of mAb Pro_006 (5.07 mg/mL) in reaction buffer (20 mM sodium phosphate, 150 mM NaCI; 20 mM EDTA, pH 7.5), was warmed to 40 °C for 15 min. TCEP (5 mM, 2 equiv. per mAb) was added to the mAb solution, mixed gently and incubated at 40 °C for 1 h. A stock solution of conjugation reagent, mc-val-cit-PAB-MMAE ( Figure 19) was prepared in DMF at 2.8 mM.
• the reduced mAb was cooled to 22 °C, diluted to 4.2 mg/mL with reaction buffer and mc-val-cit-PAB-MMAE (5.25 equiv. per mAb) was added.
• the conjugation mixture was incubated at 22 °C for 2 h.
• the crude conjugation mixture was treated with 50 mM /V-acetyl-L-cysteine (20 equiv. over reagent) at 22 °C for 30 min.
• the reaction mixture was diafiltered against DPBS using a Vivaspin20 concentrator fitted with 30 kDa MWCO PES membranes.
• the diafiltered ADC solution was buffer exchanged into DPBS using a Centripure P50 column.
• Conjugation 2 A solution of mAb Pro_006 (5.07 mg/mL) in reaction buffer (20 mM sodium phosphate 150 mM NaCI; 20 mM EDTA), pH 7.5 was warmed to 40 °C for 15 min. TCEP (5 mM, 2.75 equiv. per mAb) was added to the mAb solution, mixed gently and incubated at 40 °C for 1 h. A stock solution of conjugation reagent, mc-val-cit-PAB-MMAE ( Figure 19) was prepared in DMF at 4.0 mM.
• the reduced mAb was cooled to 22 °C, diluted to 4.2 mg/mL with reaction buffer and mc-val-cit-PAB-MMAE (7 equiv. per mAb) was added.
• the conjugation mixture was incubated at 22 °C for 2 h.
• the crude conjugation mixture was treated with 50 mM /V-acetyl-L-cysteine (20 equiv. over reagent) at 22 °C for 30 min.
• the reaction mixture was diafiltered against DPBS using a Vivaspin20 concentrator fitted with 30 kDa MWCO PES membranes.
• the diafiltered ADC solution was buffer exchanged into DPBS using a Centripure P50 column.

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