Antibodies for binding PSMA with reduced affinity for neonatal fc receptor

Abstract

To provide a modified antibody of class IgG for use in radioimmunotherapy.SOLUTION: An anti-PSMA antibody comprises a heavy chain constant region comprising one or more amino acid substitutions compared to a wild-type IgG, wherein the one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn), thereby reducing the serum half-life of the modified antibody compared to a wild-type antibody of class IgG. The one or more amino acid modifications having the effect of reducing FcRn binding are selected from positions His310, His433, His435, His436, and Ile253. The antibodies of the present invention are particularly suited for use in radioimmunotherapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies, compositions and methods for producing antibodies, particularly antibodies conjugated to radioisotopes, with reduced serum half-lives for use in radioimmunotherapy.

[0002] Related Applications This application claims priority from Australian provisional patent application Australian Patent Application Publication No. 2019902344, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Radiation therapy is an important form of tumor therapy. Various methods of radiation therapy have been developed to treat tumors. In particular, radioimmunotherapy (RAIT) is one new approach to the delivery of radiation therapy. It uses antibodies or antibody fragments to direct radioisotopes to specific tissues and cells, thereby improving the specificity of tumor treatment and reducing toxicity. RAIT further reduces its side effects by using low-dose-rate radiation.

[0004] Radiation damage to healthy tissues and organs is a major problem associated with radiation therapy. Such damage is mainly due to radiation-generated reactive oxygen species, which oxidize functionally important biological molecules such as nucleic acids, carbohydrates, lipids and lipoproteins, damaging tissues and cells. Reactive oxygen species are involved in diverse biological processes, such as antibacterial defense, inflammation, carcinogenesis and aging. Bone marrow suppression and cytopenia, such as reduction in white blood cell (WBC) and platelet counts and hematopoietic toxicity, as reflected by weight loss, are the most notable consequences of radiation injury. This toxicity severely limits the radiation dose of RAIT and reduces the effectiveness of tumor treatment.

[0005] Many methods have been developed to try to reduce the hematopoietic toxicity of radiation. Stem cell transplantation (SCT) and bone marrow transplantation (BMT) are the most frequently used methods. However, these approaches are invasive, expensive, and may contribute to longer hospital stays for individuals undergoing treatment.

[0006] Other methods include using cytokines to stimulate the immune system and blood regulatory proteins such as HP5b to turn off hematopoiesis during radiation exposure. These methods have achieved varying degrees of success in addressing hematopoietic toxicity in small studies, but again require exposing patients to additional medications and treatments and therefore remain largely unused. Summary of the Invention [Problem to be solved by the invention]

[0007] There remains a need for new methods to reduce the toxicity associated with radioimmunotherapy. [Means for solving the problem]

[0008] The reference in this specification to any prior art is not intended to be construed as forming part of the common general knowledge in any jurisdiction, or as being reasonably likely to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. It is not an admission or suggestion of what may be expected.

[0009] The present invention provides modified antibodies of class IgG for use in radioimmunotherapy, the antibodies comprising a heavy chain constant region having one or more amino acid substitutions compared to a wild-type antibody of class IgG, the one or more amino acid substitutions reducing the affinity of the antibody for the neonatal Fc receptor (FcRn) and thereby reducing the serum half-life of the modified antibody compared to a wild-type antibody of class IgG.

[0010] In one embodiment, the one or more amino acid substitutions are selected from substitutions in the heavy chain constant region 2 (CH2) of the IgG molecule, which reduces the affinity of the IgG molecule for FcRn. Alternatively, the one or more amino acid substitutions may be present in the heavy chain constant region 3 (CH3) of the IgG molecule, which reduces the affinity of the IgG molecule for FcRn. Still further, the amino acid substitutions may include at least one substitution in the CH2 region and at least one substitution in the CH3 region of the IgG molecule, which reduces the affinity of the IgG molecule for FcRn.

[0011] In certain preferred embodiments, the one or more amino acid substitutions may be at one or more of IgG residues His310, His433, His435, His436 or Ile253. Preferably, the amino acid substitution comprises a substitution at position His310 or His435 in the heavy chain constant region. More preferably, the amino acid substitution that reduces the affinity of the antibody for FcRn is at both His310 and His435.

[0012] In certain embodiments, the modified antibodies retain the ability to bind to one or more Fc-gamma receptors and thus, in certain embodiments, the modified antibodies retain the ability to stimulate an effector response (including ADCC).

[0013] In alternative embodiments, the one or more amino acid modifications that reduce affinity for the FcRn receptor also reduce affinity for an Fc gamma receptor. The modified antibody may further comprise one or more amino acid substitutions compared to a wild-type antibody of class IgG, where the amino acid substitutions further reduce the affinity of the antibody for one or more Fc gamma receptors.

[0014] In a further embodiment, the modified antibody further comprises one or more amino acid substitutions compared to a wild-type antibody of class IgG, wherein the amino acid substitutions increase the stability of the CH1-CH2 hinge region in the modified antibody compared to a wild-type antibody of class IgG.

[0015] In one embodiment, the modified antibody is conjugated to a diagnostic or therapeutic agent. The diagnostic or therapeutic agent may be directly or indirectly conjugated to the antibody, for example, by halogenation of an amino acid residue. Preferably, the diagnostic or therapeutic agent is indirectly conjugated to the antibody by a linker or chelator moiety. In one example, the modified antibody is conjugated to a chelating moiety selected from the group consisting of: TMT (6,6″-bis[N,N′′,N′″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-N-N′,N″(N′″-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, Diamsar, DTPA, CHX-A″-DTPA, TETE, Te2A, HBED, DFO, DFOsq, and HOPO or other chelating agents described herein.

[0016] In another example, the modified antibody is conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or using any amine or thiol-modifying chemistry known in the art. do.

[0017] Preferably, the diagnostic or therapeutic agent is a radioisotope. Examples of suitable isotopes include actinium-225 ( 225 Ac), astatine-211( 211 At), Bismuth-212 and Bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), Gallium-67 and Gallium-68 ( 67 Ga and 68 Ga), Indium-111 ( 111 In), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212(212 Pb), Lutetium-177( 177 Lu), Radium-223 ( 223 Ra), Samarium-153( 153 Sm), Scandium-44 and Scandium-47 ( 44 Sc, 47 Sc), Strontium-90 ( 90 Sr), Technetium-99 ( 99m Tc), yttrium-86 and yttrium-90 ( 86 Y, 90 Y), Zirconium-89( 89 Zr).

[0018] The class IgG modified antibody having reduced FcRn binding affinity compared to an unmodified antibody of class IgG can be any antibody useful for targeting diagnostic or therapeutic agents to a biological site. The antibody can be of any IgG class, including IgG1 (human or murine), IgG2, IgG4, murine IgG2a. In a preferred example, the antibody is any antibody useful for targeting or delivering diagnostic or therapeutic agents to cancer cells. Examples of suitable antibodies include the IgG1 antibodies trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab (Avastin®), dinutuximab (Unituxin®), the IgG2 antibody panitumumab (Vectibix®), the IgG4 antibodies pembrolizumab (Keytruda®), nivolumab (Opdivo®), the murine IgG2a antibody tositumomab (Bexxar®), and the murine IgG1 antibody ibritumomab (Zevalin®). Other examples include gemtuzumab (Mylotarg®), brentuximab (Adcetris®), inotuzumab (Besponsa®), glenbatumumab (CDX-011), anetumab (BAY 94-9343), mirvetuximab (IMGN853), depatuxizumab (ABT-414), rovalpituzumab (Rova-T), and rovalpituzumabutariline (SGN-CD33A).

[0019] The present invention also provides modified antibodies of class IgG having reduced FcRn binding affinity compared to an unmodified antibody of class IgG or compared to a wild-type antibody of class IgG, the antibody comprising: - a heavy chain constant region having one or more amino acid substitutions compared to a wild-type antibody of class IgG, which one or more amino acid substitutions reduce the affinity of the antibody for the neonatal Fc receptor (FcRn) and thereby reduce the serum half-life of the engineered antibody compared to a wild-type antibody of class IgG. Including, The antibody specifically binds to prostate specific membrane antigen (PSMA), the antibody comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequence of any of the complementarity determining regions has an amino acid sequence as set forth in Table 1 below. Preferably, the framework regions have an amino acid sequence as set forth in Table 1 below, containing amino acid modifications at specific residues that can be determined by aligning various framework regions from each antibody. The present invention also includes those in which CDR1, CDR2 and CDR3 are sequences from VH and CDR1a, CDR2a and CDR3a are sequences from VL, or those in which CDR1, CDR2 and CDR3 are sequences from VL and CDR1a, CDR2a and CDR3a are sequences from VH.

[0020] More particularly, the present invention provides modified antibodies of class IgG having reduced FcRn binding affinity compared to a non-modified antibody of class IgG or compared to a wild-type antibody of class IgG, the antibodies comprising: a heavy chain constant region, in which one or more amino acid residues at positions His310, His433, His435, His436, Ile253 differ from those present in an unmodified antibody or a wild-type antibody of class IgG; Including, The antibody specifically binds to prostate specific membrane antigen (PSMA), the antibody comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequence of any of the complementarity determining regions has an amino acid sequence as set out in Table 1 below. Preferably, the framework regions have an amino acid sequence as set out in Table 1 below as well, including amino acid modifications at specific residues that can be determined by aligning the various framework regions from each antibody. The present invention also includes those in which CDR1, CDR2 and CDR3 are sequences from VH and CDR1a, CDR2a and CDR3a are sequences from VL, or CDR1, CDR2 and CDR3 are sequences from VL and CDR1a, CDR2a and CDR3a are sequences from VH.

[0021] In one embodiment, an antibody that specifically binds to PSMA comprises an antigen binding site that consists essentially of or consists of the amino acids of SEQ ID NO: 4 or 20 (in N-to-C-terminal or C-to-N-terminal order).

[0022] In further embodiments, the antibody that specifically binds to PSMA comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 17, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2 or 18, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 3 or 19; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4 or 20; (iii) CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 33, CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 34, and at least about 80%, at least 85%, at least 90%, at least 92% identical to the sequence set forth in SEQ ID NO: 35; a VL comprising a CDR3 comprising a sequence at least 95%, at least 97%, at least 99% identical; (iv) a VL comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 36; (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; (vi) a VH comprising a sequence as set forth in SEQ ID NO: 4 or 20; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 33, a CDR2 comprising the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the sequence set forth in SEQ ID NO: 45; (viii) a VL comprising the sequence set forth in SEQ ID NO: 36; (ix) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 33, a CDR2 comprising the sequence shown in SEQ ID NO: 34, and a CDR3 comprising the sequence shown in SEQ ID NO: 35; or (x) a VH comprising the sequence shown in SEQ ID NO: 4 or 20 and a VL comprising the sequence shown in SEQ ID NO: 36.

[0023] Preferably, the heavy chain constant region contains amino acid substitutions at both His310 and His435. The antibody may also contain amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region.

[0024] In any embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 49-51, and preferably, the heavy chain constant region comprises the sequence set forth in SEQ ID NO: 50.

[0025] In still further embodiments, the heavy chain of the antibody comprises the sequence set forth in any one of SEQ ID NOs: 49-56, preferably SEQ ID NO: 53.

[0026] Still further, in a preferred embodiment, the light chain constant region of the antibody comprises the sequence set forth in SEQ ID NO: 52. More preferably, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:57.

[0027] In a particularly preferred embodiment, the antibody comprises the amino acid sequence set forth in SEQ ID NO:53 and the sequence set forth in SEQ ID NO:57.

[0028] The present invention also provides a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, the immunoglobulin portion specifically binds to a tumor-associated antigen, the immunoglobulin portion has reduced or eliminated affinity for the FcRn receptor compared to a wild-type immunoglobulin; Non-protein agents include therapeutic moieties such as cytotoxins or radioactive elements.

[0029] The immunoglobulin moiety is a class of therapeutic agent that may be used in the treatment of cancer, including trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab (Avastin®), dinutuximab (Unituxin®), panitumumab (Vectibix®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), tositumomab (Bexxar®), ibritumomab (Zevalin®), gemtuzumab (Mylotarg®), brentuximab (Adcetris®), inotuzumab (Besponsa®), and pembrolizumab (Keytruda®). (R), glenbatumumab (CDX-011), anetumab (BAY 94-9343), mirvetuximab (IMGN853), depatuxizumab (ABT-414), rovalpituzumab (Rova-T), and rovalpituzumabutariline (SGN-CD33A), or any other antibody described herein.

[0030] The present invention provides a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, the immunoglobulin portion specifically binds to PSMA; and FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a wherein the antigen binding site comprises FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a and CDR3a are each a complementarity determining region; Any of the sequences of the complementarity determining regions has an amino acid sequence as set forth in Table 1 below, the immunoglobulin portion has reduced or eliminated affinity for the FcRn receptor compared to a wild-type immunoglobulin; Non-protein agents include therapeutic moieties such as cytotoxins or radioactive elements.

[0031] Preferably, the framework regions have the amino acid sequences also set out in Table 1 below, including amino acid modifications at specific residues that can be determined by aligning the various framework regions from each antibody. The invention also includes those in which CDR1, CDR2 and CDR3 are sequences from VH and CDR1a, CDR2a and CDR3a are sequences from VL, or CDR1, CDR2 and CDR3 are sequences from VL and CDR1a, CDR2a and CDR3a are sequences from VH. Preferably, the immunoglobulin portion has amino acid substitutions at residues equivalent to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also include amino acid substitutions at residues equivalent to Ser228 and Leu235 in the constant heavy chain region.

[0032] Preferably, the non-protein agent comprises a radioactive element.

[0033] The present invention also provides a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, the immunoglobulin portion specifically binds to PSMA and comprises an antigen-binding site that consists essentially of or consists of the amino acids of SEQ ID NO: 4 or 20 (in N-to-C-terminal or C-to-N-terminal order); the immunoglobulin portion has reduced or eliminated affinity for the FcRn receptor compared to a wild-type immunoglobulin; Non-protein agents include therapeutic moieties such as cytotoxins or radioactive elements.

[0034] Preferably, the immunoglobulin portion has amino acid substitutions at residues equivalent to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also include amino acid substitutions at residues equivalent to Ser228 and Leu235 in the constant heavy chain region.

[0035] The present invention also provides a molecule comprising an immunoglobulin portion and a non-protein agent conjugated thereto, the immunoglobulin portion has reduced or eliminated affinity for the FcRn receptor compared to a wild-type immunoglobulin, the immunoglobulin portion specifically binds to PSMA, and Contains at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 17, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2 or 18, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 3 or 19; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4 or 20; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 33, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 35; (iv) a VL comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 36; (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; (vi) a VH comprising a sequence as set forth in SEQ ID NO: 4 or 20; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 33, a CDR2 comprising the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the sequence set forth in SEQ ID NO: 45; (viii) a VL comprising the sequence set forth in SEQ ID NO: 36; (ix) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 33, a CDR2 comprising the sequence shown in SEQ ID NO: 34, and a CDR3 comprising the sequence shown in SEQ ID NO: 35; or (x) a VH comprising the sequence shown in SEQ ID NO: 4 or 20 and a VL comprising the sequence shown in SEQ ID NO: 36.

[0036] Preferably, the immunoglobulin portion has amino acid substitutions at residues equivalent to His310 and / or His435 in the constant heavy chain region. The immunoglobulin portion may also include amino acid substitutions at residues equivalent to Ser228 and Leu235 in the constant heavy chain region. Preferably, the non-protein agent comprises a radioactive element.

[0037] In any embodiment, the immunoglobulin comprises a heavy chain constant region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 49-51, and preferably, the heavy chain constant region comprises the sequence set forth in SEQ ID NO: 50.

[0038] In still further embodiments, the immunoglobulin comprises the sequence shown in any one of SEQ ID NOs: 53-56, preferably SEQ ID NO: 53.

[0039] In any embodiment, the immunoglobulin comprises a light chain constant region comprising the sequence of SEQ ID NO: 52. In one embodiment, the immunoglobulin comprises a light chain having the amino acid sequence shown in SEQ ID NO:57.

[0040] In a particularly preferred embodiment, the immunoglobulin comprises the sequences shown in SEQ ID NOs:53 and 57.

[0041] The present invention also provides a method of treating cancer in an individual, the method comprising administering to an individual in need thereof a molecule comprising an immunoglobulin portion and a non-proteinaceous agent conjugated as described above.

[0042] The present invention also provides a method for producing an antibody suitable for use in radioimmunotherapy, the method comprising: - providing an antibody having an antigen binding site that specifically binds to an epitope present on a cell or tissue in need of radioimmunotherapy; - introducing at least one amino acid substitution in the heavy chain constant region of the antibody to produce an altered antibody, wherein the at least one amino acid substitution is selected from the group consisting of amino acid substitutions at residues His310, His435 and Ile253, thereby causing an alteration of the binding affinity to FcRn and / or the serum half-life of said antibody; - conjugating the modified antibody with a radioactive element thereby producing antibodies suitable for use in radioimmunotherapy.

[0043] In certain embodiments, the antibody is an antibody described herein, including antibodies having any of the complementarity determining regions, framework regions, variable light chain or variable heavy chain regions set forth in Table 1 below. Preferably, the modified antibody also contains amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region. Preferably, the radioactive element is conjugated to the modified antibody using a chelating agent, such as DOTA.

[0044] The invention also provides a method of producing an antibody-radioisotope immunoconjugate for use in the treatment of disease, the method comprising: - providing an antibody having an antigen binding site that specifically binds to an epitope present on a cell or tissue in need of radioimmunotherapy; - introducing at least one amino acid substitution in the heavy chain constant region of the antibody to produce an altered antibody, wherein the at least one amino acid substitution is selected from the group consisting of substitutions at amino acid residues His310, His435 and Ile253, thereby causing an alteration of the binding affinity to FcRn and / or the serum half-life of said antibody; - conjugating the modified antibody with a radioactive element thereby producing an antibody-radioisotope immunoconjugate for use in the treatment of disease.

[0045] Preferably, the antibody is an antibody as described herein, including antibodies having any of the complementarity determining regions, framework regions, variable light chain or variable heavy chain regions set out in Table 1 below. Preferably, the modified antibody also comprises amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region, and preferably the radioactive element is conjugated to the modified antibody using a chelating agent, such as DOTA. More preferably, the modified antibody comprises the amino acid substitutions His310Ala and His435Gln.

[0046] Preferably, the disease is cancer, including prostate cancer or renal cell carcinoma.

[0047] The invention further provides a method for producing an engineered antibody having an altered binding affinity for FcRn and / or an altered serum half-life compared to an unmodified form of the antibody, the method comprising: (a) an expression vector (preferably a replicable expression vector) comprising a suitable promoter operably linked to a nucleic acid molecule encoding at least a constant region of an immunoglobulin heavy chain; wherein at least one amino acid from the heavy chain constant region selected from the group consisting of amino acid residues His310, His435, and Ile253 is replaced with an amino acid different from that present in the unmodified antibody, thereby causing an alteration in FcRn binding affinity and / or serum half-life; (b) transforming a host cell with the vector; (c) culturing the transformed host cell to produce the modified antibody; Includes.

[0048] Optionally, such methods further comprise preparing a second expression vector (preferably a replicable expression vector) comprising a promoter operably linked to DNA encoding a complementary immunoglobulin light chain, and further transforming the cell line with said second vector.

[0049] 1. A method for altering the serum half-life of an antibody for use in radioimmunotherapy, comprising: - providing an antibody having an antigen binding site that specifically binds to an epitope present on a cell or tissue in need of radioimmunotherapy; - introducing at least one amino acid substitution into the heavy chain constant region of the antibody to produce an altered antibody, wherein the at least one amino acid substitution is selected from the group consisting of substitutions at amino acid residues His310, His435 and Ile253, thereby causing a change in the binding affinity to FcRn and the serum half-life of the antibody. The method includes:

[0050] Preferably, the method further comprises conjugating the modified antibody with a radioactive element. Preferably, the antibody is an antibody as described herein for binding to PSMA, including an antibody having any of the complementarity determining regions, framework regions, variable light chain or variable heavy chain regions set forth in Table 1 below. Preferably, the modified antibody also comprises amino acid substitutions at residues equivalent to Ser228 and Leu235 of the constant heavy chain region, including Ser228Pro and / or Leu235Glu. More preferably, the modified antibody comprises the amino acid substitutions His310Ala and His435Gln.

[0051] 1. A method for reducing the toxicity of an antibody for use in radioimmunotherapy, comprising: - providing an antibody having an antigen binding site that specifically binds to an epitope present on a cell or tissue in need of radioimmunotherapy; - introducing at least one amino acid substitution into a heavy chain constant region of the antibody to produce a modified antibody, wherein the at least one amino acid substitution is selected from the group consisting of substitutions at amino acid residues His310, His435 and Ile253, and the amino acid substitution reduces the serum half-life of the modified antibody and / or increases the clearance of the modified antibody from circulation. thereby reducing the toxicity of antibodies when they are conjugated with radioactive elements for use in radioimmunotherapy.

[0052] In any embodiment, reducing the toxicity of the antibody includes reducing many of the toxic effects that would otherwise result from longer residence of the radioisotope in the circulation, including hematological toxicity, absorption into the bone, and bone marrow irradiation.

[0053] In any embodiment, the toxicity of a radiolabeled antibody or radioimmunoconjugate described herein is assessed by determining the tumor:blood ratio of the antibody or immunoconjugate following administration to an individual.

[0054] In any embodiment of the invention, the tumor:blood ratio of an engineered antibody of the invention is at least 2-fold, at least 3-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold greater than an unmodified antibody that does not have the modifications to the heavy chain constant region described herein, when the ratio is determined at least 8 hours after administration of the antibody. Alternatively, the ratio is determined at least 24, 48, 72, or 120 hours after administration of the antibody to the individual. In certain embodiments, the tumor:blood ratio of an engineered antibody of the invention is at least 50-fold, at least 100-fold, at least 200-fold, or at least 300-fold greater than an unmodified antibody that does not have the modifications to the heavy chain constant region described herein, when the ratio is determined at least 120 hours after administration of the antibody.

[0055] In any embodiment of the invention, the modified antibodies described herein that have a reduced or altered serum half-life compared to the unmodified antibody have a serum clearance rate that is at least two-fold, at least three-fold faster or more than the unmodified antibody.

[0056] In a particularly preferred embodiment of the invention, the antibodies described herein are suitable for use in a theranostic couple, where the theranostic couple comprises 1) an antibody conjugated to an imaging agent, and 2) an antibody conjugated to a therapeutic agent. For example, the antibodies may be used first as diagnostic agents when conjugated to a radioisotope suitable for use in radioimaging, and secondly, the antibodies may be used as therapeutic agents when conjugated to a radioisotope or cytotoxic agent suitable for use in therapy.

[0057] The present invention also provides an in vivo method of diagnosing, monitoring or predicting a disease, disorder or infection in a subject, the method comprising: (a) administering to a subject an effective amount of an engineered antibody described herein, wherein the engineered antibody specifically binds to an antigen associated with a disease, disorder, or infection; (b) concentrating the engineered antibodies at sites in the subject where the antigen is found; (c) detecting the modified antibody; and whereby detection of said modified antibody above background or standard levels indicates that the subject has said disease, disorder or infection.

[0058] The present invention provides antigen-binding sites which bind or specifically bind to prostate-specific membrane antigen (PSMA). Preferably, the antigen-binding sites of the invention bind or specifically bind to human PSMA.

[0059] The present invention provides an antigen-binding site for binding to PSMA, the antigen-binding site comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequences of either the framework regions or the complementarity determining regions are provided herein.

[0060] The present invention provides an antigen-binding site for binding to PSMA, the antigen-binding site comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequence of any of the complementarity determining regions has an amino acid sequence as set out in Table 1 below. Preferably, the framework regions have an amino acid sequence as set out in Table 1 below as well, including amino acid modifications at specific residues that can be determined by aligning the various framework regions from each antibody. The present invention also includes those in which CDR1, CDR2 and CDR3 are sequences from VH and CDR1a, CDR2a and CDR3a are sequences from VL, or CDR1, CDR2 and CDR3 are sequences from VL and CDR1a, CDR2a and CDR3a are sequences from VH.

[0061] The present invention provides an antigen binding site comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 4 or 20 (in N to C-terminal or C to N-terminal order).

[0062] The invention also provides an antigen-binding site comprising an antigen-binding domain of an antibody, wherein the antigen-binding domain binds or specifically binds to PSMA, and wherein the antigen-binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 17, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2 or 18, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 3 or 19; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4 or 20; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 33, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 35; (iv) a VL comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 36; (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; (vi) a VH comprising a sequence as set forth in SEQ ID NO: 4 or 20; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 33, a CDR2 comprising the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the sequence set forth in SEQ ID NO: 45; (viii) a VL comprising the sequence set forth in SEQ ID NO: 36; (ix) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1 or 17, a CDR2 comprising the sequence shown in SEQ ID NO: 2 or 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 3 or 19; and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 33, a CDR2 comprising the sequence shown in SEQ ID NO: 34, and a CDR3 comprising the sequence shown in SEQ ID NO: 35; or (x) a VH comprising the sequence shown in SEQ ID NO: 4 or 20 and a VL comprising the sequence shown in SEQ ID NO: 36.

[0063] In any aspect of the invention, the antigen binding domain further comprises at least one of the following: (i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 9 or 25, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 10 or 26, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 11 or 27, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 12 or 28; (ii) a VL comprising an FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 41, an FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 42, an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 43, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 44; (iii) VH comprising FR1 comprising the sequence shown in SEQ ID NO: 9 or 25, FR2 comprising the sequence shown in SEQ ID NO: 10 or 26, FR3 comprising the sequence shown in SEQ ID NO: 11 or 27, and FR4 comprising the sequence shown in SEQ ID NO: 12 or 28; (iv) a VL comprising an FR1 comprising the sequence shown in SEQ ID NO: 41, an FR2 comprising the sequence shown in SEQ ID NO: 42, an FR3 comprising the sequence shown in SEQ ID NO: 43, and an FR4 comprising the sequence shown in SEQ ID NO: 44; or (v) a VH comprising an FR1 comprising the sequence shown in SEQ ID NO: 9 or 25, an FR2 comprising the sequence shown between SEQ ID NO: 10 or 26, an FR3 comprising the sequence shown between SEQ ID NO: 11 or 27, and an FR4 comprising the sequence shown between SEQ ID NO: 12 or 28; and a VL comprising an FR1 comprising the sequence shown in SEQ ID NO: 41, an FR2 comprising the sequence shown between SEQ ID NO: 42, an FR3 comprising the sequence shown in SEQ ID NO: 43, and an FR4 comprising the sequence shown in SEQ ID NO: 44.

[0064] In any embodiment, the antigen-binding site comprises a heavy chain constant region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 49 to 51, and preferably, the heavy chain constant region comprises the sequence set forth in SEQ ID NO: 50.

[0065] In a still further embodiment, the antigen binding site comprises the sequence shown in any one of SEQ ID NOs: 53 to 56, preferably 53.

[0066] In any embodiment, the antigen binding site comprises a light chain constant region comprising the sequence of SEQ ID NO: 52. In one embodiment, the light chain of the antigen binding site comprises the sequence of SEQ ID NO: 57.

[0067] In a particularly preferred embodiment, the antigen binding site comprises the sequences shown in SEQ ID NOs:53 and 57.

[0068] The antigen-binding site may also be referred to as an antigen-binding domain of an antibody.

[0069] Preferably, the antigen-binding site described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding site is an antibody, such as a monoclonal antibody.

[0070] As described herein, the antigen binding site may be of the following form: (i) Single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) one of (i) or (ii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (iv) one of (i) or (ii) bound to a protein that binds to an immune effector cell.

[0071] Furthermore, as described herein, the antigen binding site may be of the form: (i) Diabody; (ii) triabody; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) one of (i) to (vi) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (viii) One of (i) to (vi) bound to a protein that binds to immune effector cells.

[0072] In any aspect or embodiment, the antibody is a naked antibody. In particular, the antibody is in unconjugated form and has not been adapted to form a conjugate.

[0073] The present invention also provides fusion proteins comprising an antigen binding site, immunoglobulin variable domain, antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule or multispecific antibody as described herein.

[0074] The invention also provides conjugates in the form of antigen binding sites, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules or multispecific antibodies or fusion proteins as described herein conjugated to a label or a cytotoxic agent.

[0075] The invention also provides antibodies for binding to the antigen binding site, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule or multispecific antibody, fusion protein or conjugate described herein.

[0076] In a preferred embodiment, the antigen-binding site is an IgG immunoglobulin that comprises one or more amino acid substitutions in the antibody constant domain, CH2-CH3 region, that modify binding of the antibody to the neonatal Fc receptor (FcRn) relative to a wild-type antibody Fc region. The one or more amino acid modifications alter the affinity of the antibody constant domain, Fc region, or FcRn-binding fragment thereof, for FcRn, thereby altering the serum half-life of the antigen-binding site.

[0077] Preferably, the substitutions alter the binding affinity to FcRn and / or the serum half-life of the modified antibody relative to an unmodified wild-type antibody. The invention further provides modified antibodies having reduced binding affinity to FcRn and / or reduced serum half-life compared to the unmodified antibody, wherein any one or more amino acid residues at positions Ile253 or His310 from the CH2 domain and / or residue His435 from the CH3 domain are replaced with another amino acid that is different to that present in the unmodified antibody or different from the unmodified IgG.

[0078] In one example, the one or more amino acid modifications are selected from amino acid substitutions at residues equivalent to H310 and H435. In a further example, the antibody includes amino acid substitutions at both the His310 and His435 residues.

[0079] The amino acid substitution may include a substitution of a histidine residue with alanine, glutamine, glutamic acid or aspartic acid. Preferably, the amino acid substitution at His310 is with alanine. Preferably, the amino acid substitution at His435 is with glutamine. Preferably, the amino acid substitution at Ile253 is with alanine.

[0080] In a further embodiment, the antigen-binding site is an antibody comprising one or more amino acid substitutions that alter the binding of the antibody to activate Fc gamma receptors. The one or more amino acid modifications alter the affinity of the antibody constant domain, Fc region or Fc gamma receptor binding fragment to any one or more Fc gamma receptors. Preferably, the amino acid modification is at the residue equivalent to Leu235. More preferably, the amino acid modification is from Leu235 to glutamic acid.

[0081] In one embodiment, the amino acid modification is a hinge stabilizing mutation at Ser228. Preferably, the amino acid modification at Ser228 is to proline.

[0082] In one embodiment of the invention, the antibody comprises mutations at Ser228, Leu235, His310 and His435. Preferably, the amino acid modifications are Ser228Pro, Leu235Glu, His310Ala and His435Gln.

[0083] The amino acid modifications are preferably made to antibodies having an IgG1 isotype or an IgG4 isotype.

[0084] In a preferred embodiment, the antibody comprises a heavy chain constant region shown in any one of SEQ ID NOs:235-238.

[0085] The invention also provides nucleic acids encoding an antigen binding site, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule or multispecific antibody, fusion protein or conjugate described herein.

[0086] In one example, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be in a vector, such as a plasmid.

[0087] In an embodiment of the invention relating to a single polypeptide chain antigen binding site, the expression construct may include a promoter linked to a nucleic acid encoding the polypeptide chain.

[0088] In the example of multiple polypeptide chains forming an antigen binding site, the expression construct Examples of such nucleic acids include a nucleic acid encoding a polypeptide comprising, for example, a VH operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising, for example, a VL operably linked to a promoter.

[0089] In another example, the expression construct is a bicistronic expression construct that includes, for example, the following operably linked components in 5' to 3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide; Here, the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0090] The present invention also contemplates separate expression constructs, one encoding a first polypeptide comprising a VH and the other encoding a second polypeptide comprising a VL. For example, the present invention also provides a composition comprising: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0091] The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises an expression construct of the invention or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; wherein the first and second polypeptides combine to form the antigen-binding site of the invention.

[0092] Examples of cells of the invention include fungal cells, yeast cells, insect cells or mammalian cells.

[0093] The present invention also provides a pharmaceutical composition comprising an antigen binding site or comprising the CDR and / or FR sequences described herein or an immunoglobulin variable domain as described herein, an antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule or multispecific antibody, fusion protein or conjugate and a pharma- ceutically acceptable carrier, diluent or excipient.

[0094] The present invention also provides a diagnostic composition comprising an antigen binding site or comprising the CDR and / or FR sequences described herein or the antigen binding site described herein, an immunoglobulin variable domain, an antibody, a dab, a di-scFv, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate, a diluent and optionally a label. Preferably, the antigen binding site is a monoclonal antibody conjugated to a radioisotope.

[0095] The present invention relates to an immunoglobulin variable domain, antibody, dab, di-sc comprising an antigen binding site or CDR and / or FR sequences as described herein or an immunoglobulin variable domain as described herein. Kits or articles of manufacture comprising the Fv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule or multispecific antibody, fusion protein or conjugate are also provided.

[0096] Preferably, the antigen-binding moiety is a monoclonal antibody conjugated to a radioisotope.

[0097] The antigen-binding site, protein or antibody described herein may comprise a human constant region, such as an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region or a mixture thereof. When an antibody or protein comprises a VH and a VL, the VH may bind to a heavy chain constant region and the VL may bind to a light chain constant region.

[0098] In one example, a protein or antibody described herein, or a composition of a protein or antibody described herein, comprises a heavy chain constant region that comprises a stabilized heavy chain constant region that comprises a mixture of sequences with or without a C-terminal lysine residue, either completely or partially.

[0099] In one example, an antibody of the invention comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as described above), and the VL is linked or fused to a kappa light chain constant region.

[0100] The functional properties of the antigen binding sites of the present invention will be understood to apply mutatis mutandis to the antibodies of the present invention.

[0101] The antigen binding sites described herein may be purified, substantially purified, isolated and / or recombinant.

[0102] The invention also provides a method of treating or preventing cancer in a subject, the method comprising administering an antigen binding site of the invention to the subject. In this regard, the antigen binding site may be used to prevent the recurrence of the condition, which is considered prevention of the condition.

[0103] Exemplary cancers include prostate cancer. It will be appreciated that antibodies with affinity for PSMA are useful for this purpose.

[0104] The present invention also provides an in vivo method of diagnosing, monitoring or predicting a disease, disorder or infection in a subject, the method comprising: (a) administering to a subject an effective amount of an antibody described herein, wherein the antibody specifically binds to an antigen associated with the disease, disorder, or infection; (b) concentrating antibodies at sites in the subject where the antigen is found; (c) detecting the antibody; whereby detection of said antibody above background or standard levels indicates that the subject has said disease, disorder or infection.

[0105] As used herein, unless the context otherwise requires, the term "comprise" and variations of the term, such as "comprises", "comprises" and "comprised", are not intended to exclude further appended elements, components, integers or steps.

[0106] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0107] [Figure 1] Mean half-life and Tukey's multiple comparison of half-lives of antibodies of the invention. Error bars represent standard error of the mean. J591 IgG = control HuJ591 antibody for PSMA binding. ANT4044-K-DOTA = antibody ANT4044 ricin conjugated to DOTA. ANT4044-A2-K-DOTA- antibody ANT4044-A2 ricin conjugated to DOTA. ANT4044-FcRn-K-DOTA = antibody ANT4044 with amino acid substitutions in the FcRn binding region, ricin conjugated to DOTA. ANT4044-FcRg-K-DOTA = antibody ANT4044 with amino acid substitutions in the FcRn and Fc gamma receptor binding region, ricin conjugated to DOTA. [Diagram 2]Mean area under the curve (AUC) and clearance (CL) for selected antibodies of the invention. J591 IgG = control HuJ591 antibody for PSMA binding. ANT4044-K-DOTA = antibody ANT4044 ricin conjugated to DOTA. ANT4044-A2-K-DOTA-antibody ANT4044-A2 ricin conjugated to DOTA. ANT4044-FcRn-K-DOTA = antibody ANT4044 with amino acid substitutions in the FcRn binding region, ricin conjugated to DOTA. ANT4044-FcRg-K-DOTA = antibody ANT4044 with amino acid substitutions in the FcRn and Fc gamma receptor binding regions, ricin conjugated to DOTA. [Diagram 3] Antibody biodistribution at 8 hours determined by ROI analysis of PET images. [Figure 4] Antibody biodistribution at 24 hours determined by ROI analysis of PET images. [Diagram 5] Total antibody biodistribution at 48 hours as determined by ex vivo gamma counting. [Figure 6] Antibody biodistribution at 48 hours determined by ROI analysis of PET images. [Figure 7] Antibody blood levels up to 5 days after injection. [Figure 8] Tumor accumulation of antibody determined by imaging (8 h, 24 h, 48 h). [Figure 9] Antibody in tumor to blood ratio. The tumor:blood ratio (tumor:tail blood in vivo) for each of the antibodies was determined for the 8, 24 and 48 hour time points. Compared to antibodies JN005 and hJ591, the ratios for JN006 and JN007 are significantly higher at all time points. [Figure 10] Antibody in tumor to blood ratio. The tumor:blood ratio (ex vivo:ex vivo) for each of the antibodies was determined for the 48 hour and 120 hour time points. Compared to antibodies JN005 and hJ591, the ratios for JN006 and JN007 are significantly higher at all time points. [Figure 11]In vivo imaging and in vivo distribution of an exemplary antibody of the invention. SPECT imaging of LNCap xenograft mice receiving an anti-PSMA, FcRn-K-DOTA-Lu-engineered antibody of the invention. [Figure 12] Blood pharmacokinetics of an exemplary antibody of the invention. Levels of radioactivity measured in the blood of mice following administration of an anti-PSMA, FcRn-K-DOTA-Lu-engineered antibody of the invention. [Figure 13] Efficacy study in LNCap-bearing xenograft mice treated with exemplary antibodies of the invention. Treatment with the anti-PSMA, K-DOTA-Lu FcRn-modified antibodies of the invention significantly inhibited tumor growth as evidenced by no change in tumor volume at day 14 compared to day 0. In the control (PBS) group, there was an overall increase in tumor volume, with tumors becoming significantly larger at days 9, 12, and 14 when compared to the corresponding times in the FcRn-K-DOTA-Lu treated group. [Figure 14] Tumor:blood ratios in LNCap-bearing xenografted mice after administration of an exemplary antibody of the invention. Tumor:blood ratios are shown for mice treated with an anti-PSMA, K-DOTA-Lu antibody of the invention modified to reduce FcRn binding (HuX592R-DOTA-Lu177). Control mice were administered an anti-PSMA, K-DOTA-Lu antibody (HuJ591-DOTA-Lu177). Ratios are greater in mice that received the FcRn modified antibody compared to mice that received the unmodified antibody, especially at 24 and 48 hours. [Figure 15] 177Lu-labeled HuX592R and HuJ591 biodistribution in healthy male Balb / c nude mice as measured by ex vivo gamma counting. A shows HuX592R biodistribution assessed at 24, 48 and 72 hours post-injection, while B compares HuX592R biodistribution with HuJ591 at 72 hours post-injection. [Figure 16] Regression of LNCaP xenograft tumors in each treatment cohort following administration of HuX592R(FcRn-K-DOTA-Lu) or untreated controls. [Figure 17]Plot of cohort survival over the study period following administration of TLX592 (FcRn-K-DOTA-Lu), TLX591 (K-DOTA-Lu antibody, also referred to herein as HuJ591-DOTA-Lu177), or untreated control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0109] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings, in which:

[0110] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

[0111] The present invention relates, in part, to the identification of new approaches for reducing the toxicity of radioimmunoconjugates for use in radioimmunotherapy. In particular, the methods of the present invention reduce toxicity without significantly affecting the therapeutic potential of the radioimmunoconjugates.

[0112] As outlined above, radiation damage to healthy tissues and cells is a major problem associated with radioimmunotherapy. This toxicity severely limits the radiation dose of RAIT and reduces the effectiveness of tumor treatment.

[0113] However, the present inventors have developed an antibody for use in radioimmunotherapy that has a reduced serum half-life compared to the wild-type antibody by reducing the affinity of the antibody for the neonatal Fc receptor (FcRn) through modifications to the constant heavy chain of the antibody. Thus, the antibody developed by the present inventors has significant advantages for use in various immunotherapies.

[0114] Modifications of the FcRn-binding domain of therapeutic antibodies have been previously reported, but such modifications were developed with the aim of increasing FcRn affinity, e.g., increasing the serum half-life of the therapeutic antibody and prolonging its residence in the circulation. In contrast to prior art approaches, the present invention aims to decrease the serum half-life of the therapeutic agent.

[0115] Surprisingly, the inventors have found that despite the reduced serum half-life (and increased rate of clearance from the systemic circulation following administration), the antibodies of the invention have a similar ability to be delivered to and accumulate at the tumor site as unmodified antibodies. These results are surprising in that they show that the tumor burden of modified and unmodified antibodies is not statistically different. and show that the approach taken by the inventors significantly reduces serum half-life and therefore toxicity, while not negatively impacting the ability of the antibodies to bind their target epitope or to be delivered to the target site.

[0116] More importantly, the inventors have shown that the modified antibodies of the present invention continue to reside at the tumor site, despite increased clearance. Thus, the inventors' studies show that modification of the FcRn or FcRn and Fc gamma receptor binding domains of radiolabeled antibodies has significant utility in reducing the amount of radioisotope in circulation, without affecting the therapeutic potential of the antibody with respect to its ability to accumulate in tumors. This has many advantages, including reducing many toxic effects that would otherwise result from longer residence of radioisotopes in circulation (including hematologic toxicity as a result of bone marrow irradiation and bone resorption). Furthermore, considering that the dose-limiting toxicity of many previous RAIT therapies is hematologic toxicity as a direct result of this prolonged blood circulation and bone marrow irradiation, the present invention offers the possibility of acceptable dosing at higher levels; thus resulting in more effective treatment (bone resorption and bone marrow irradiation).

[0117] Unexpectedly, the inventors also found that amino acid modifications to the constant heavy chain, while preventing FcRn binding, do not affect the ability of the antibody to bind to protein G and some protein A purification resins. Thus, the antibodies of the present invention, which have reduced serum half-lives compared to other immunotherapeutics, can be produced using the same existing / standardized production platforms developed for conventional antibodies. This is an important advantage over some of the many other engineered antibody formats, such as minibodies, diabodies, etc., which are cumbersome to produce and less stable than IgG molecules. Also, as a molecular format that is "native" to the body, full-length antibodies also tend to reduce the likelihood of immunogenic responses than engineered antibodies or antibody fragments.

[0118] A further advantage of the antibodies of the invention is their particular suitability for application in the field of theranostics. More specifically, as mentioned above, the reduced serum half-life of the antibodies makes them particularly useful for therapy when conjugated to a radioisotope, since they can deliver an appropriate amount of radioactive material to the tumor while being rapidly cleared from the circulation. In addition, the reduced serum half-life of the antibodies makes them particularly suitable for use in diagnostic methods, where rapid clearance of the radioisotope conjugated to the antibody and selected for imaging is desirable. The use of the antibodies in the first instance as diagnostics thereby informs the use and administration of therapeutic forms of the antibodies (i.e. when the antibodies are conjugated to a radioisotope suitable for therapy). Thus, the antibodies of the invention find utility when conjugated to different radioisotopes and subsequently used as "theranostic couples".

[0119] General matters Throughout this specification, unless otherwise stated or the context requires otherwise, a single step, composition of matter, group of steps, or group of compositions of matter is intended to include one and a plurality (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter. Thus, as used herein, the singular forms "a," "an," and "the" include plural embodiments, and vice versa, unless the context clearly dictates otherwise. For example, a reference to "a" includes the singular and more than one; a reference to "an" includes the singular and more than one; a reference to "the" includes the singular and more than one, etc.

[0120] Those skilled in the art will recognize that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention relates to the processes, features, compositions and compounds referred to or shown in this specification. This includes all and any and all combinations of said steps or features, or combinations of any two or more of said steps or features, individually or collectively.

[0121] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0122] All patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0123] The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly included within the scope of the present invention.

[0124] Any example or embodiment of the invention herein is intended to be construed as applying mutatis mutandis to any other example or embodiment of the invention, unless specifically stated otherwise.

[0125] Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0126] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are broadly based on those described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMG Lover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors), Current Protocols in Immunology, John It is described and explained throughout the literature in sources such as Wiley & Sons (including all updates to date).

[0127] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein are based on Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al. al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J.Mol Biol.196:901-917,1987, Chothia et al.Nature 342,877-883,1989 and / or Al-Lazikani et al.,J Mol Biol 273,927-94 This may be further clarified in the discussion at issue in US Pat. No. 6,313,351.

[0128] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0129] As used herein, the term "derived from" shall be interpreted as indicating that a particular integer can be obtained from a particular source, although not necessarily directly from that source.

[0130] Selected Definitions As used herein, tumor:blood ratio refers to the ratio of the amount of antibody (or radiolabeled antibody) to the amount of the same antibody in the blood of an individual. Those skilled in the art will be familiar with standard techniques for calculating tumor:blood ratio. For example, the ex vivo activity concentration of radioisotope (or labeled antibody) is measured and expressed as a percentage of decay-corrected injected activity per gram of tissue (or blood) and approximated as a percentage of injected dose / g (%ID / g). The tumor-to-blood ratio is then calculated as the activity detected in the tumor relative to the activity detected in blood.

[0131] As used herein, the term "theranostic" refers to the ability of a compound / material to be used for diagnosis and treatment. The term "theranostic reagent" relates to any reagent suitable for both detection, diagnosis and / or treatment of a disease or condition in a patient. The purpose of theranostic compounds / materials is to overcome undesirable differences in biodistribution and selectivity that may exist between different diagnostic and therapeutic agents. With theranostic couples, the theranostic compound containing an imaging radionuclide is first administered to a patient to identify the disease or locate the diseased area in the body. Once identified / localized, the disease can be treated by administering the theranostic compound containing a therapeutic radionuclide in a target-specific manner since the biodistribution of the imaging radionuclide and the therapeutic radionuclide are the same.

[0132] In the context of the present invention, the antibodies of the invention are particularly useful for inclusion in theranostic couples, for example, where an antibody is conjugated to a radioisotope for imaging or diagnostic purposes and the same antibody is conjugated to a different radioisotope or cytotoxic agent suitable for therapy. The antigen-binding site of the antibody directs or targets the diagnostic radioisotope to the site of the tumor to facilitate diagnosis (including tumor distribution, tumor size, tumor density), while the same antigen-binding site of the antibody directs the radioisotope to the tumor for therapy.

[0133] The term "Fc region" as used herein, sometimes referred to as "Fc" or "Fc domain", refers to the portion of an IgG molecule that correlates with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region consists of the C-terminal halves of the two heavy chains of an IgG molecule linked by disulfide bonds. It does not have antigen-binding activity, but contains carbohydrate moieties and binding sites for complement and Fc receptors, including the FcRn receptor. The Fc region comprises the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the EU index numbering system, also defined as residues 244-360 in the Kabat system) and the third constant domain CH3 (residues 341-447 EU index / 361-478 Kabat) (see, e.g., SEQ ID NO: 1 or FIG. 1C of WO2015175874 for the sequence of CH2 and SEQ ID NO: 2 for the sequence of CH3; FIG. 1D, which is incorporated herein by reference; also see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs for a comparison of the numbering conventions used for various residues in the Fc region of immunoglobulins).

[0134] As used herein, "EU index" or "EU numbering scheme" refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). As used herein, "Kabat system" refers to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. One of skill in the art will be able to readily determine whether a given amino acid sequence is numbered according to the EU or Kabat system.

[0135] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that is not associated with naturally associated components that accompany it in nature by its origin or source of origin; it is substantially free of other proteins from the same source. A protein can be rendered substantially free of naturally associated components by isolation, or substantially purified, using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free of contaminants, e.g., at least about 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% free of contaminants.

[0136] The term "recombinant" shall be understood to mean a product of artificial genetic recombination. Thus, in the context of a recombinant protein containing an antibody antigen-binding domain, the term does not encompass naturally occurring antibodies in a subject's body that are the product of natural recombination that occurs during B-cell maturation. However, when such an antibody is isolated, it should be considered an isolated protein that contains an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein that contains an antibody antigen-binding domain. Recombinant protein also encompasses proteins expressed by artificial recombinant means, for example, when it is in a cell, tissue, or subject in which it is expressed.

[0137] The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0138] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0139] As used herein, the term "antigen-binding site" is used interchangeably with "antigen-binding domain" and shall be taken to mean the region of an antibody capable of specifically binding to an antigen, i.e. Fv comprising VH or VL or both VH and VL. The antigen-binding domain need not be in the context of a whole antibody, e.g., isolated (e.g., domain antibody) or in another form as described herein, e.g., scFv.

[0140] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens through an antigen-binding domain contained in an Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primate antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies). Antibodies generally comprise a constant region or They contain a constant domain that can be arranged into a constant fragment or a crystallizable fragment (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain two covalently linked heavy chains (about 50-70 kDa) and two light chains (about 23 kDa each). The light chains generally contain a variable region (if present) and a constant domain, and in mammals are either kappa or lambda light chains. The heavy chains generally contain a variable region and one or two constant domains connected to further constant domains by a hinge region. Mammalian heavy chains are of one of the types α, δ, ε, γ or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids long) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids long) is aligned and disulfide-bonded with the first constant domain of the heavy chain (CH1, 330-440 amino acids long). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain may include two or more additional CH domains (e.g., CH2, CH3, etc.) and may include a hinge region between the CH1 and CH2 constant domains. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synthetic humanized, chimeric, CDR grafted or deimmunized.

[0141] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of an antibody. In particular, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0142] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody as defined herein that can specifically bind to an antigen, and includes the amino acid sequences of the complementarity determining regions (CDRs); i.e., CDR1, CDR2 and CDR3, and framework regions (FRs). For example, a variable region includes three CDRs together with three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0143] As used herein, the term "complementarity determining region" (synonyms CDR; i.e. CDR1, CDR2 and CDR3) refers to the amino acid residues of an antibody variable region, the presence of which is a major contributor to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs, identified as CDR1, CDR2 and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2 and CDR H3, respectively, where CDR H1 corresponds to CDR1 of VH, CDR H2 corresponds to CDR2 of VH and CDR H3 corresponds to CDR3 of VH. Similarly, the CDRs of VL are also referred to herein as CDR L1, CDR L2 and CDR L3, respectively, where CDR L1 corresponds to CDR1 of VL, CDR L2 corresponds to CDR2 of VL and CDR L3 corresponds to CDR3 of VL. In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the CDRs and FRs are defined according to the extended Chothia numbering scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system. The numbering system includes any numbering system including the standard numbering system or the numbering system of Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; the numbering system of Honnegher and Pluekthun J. Mol. Biol. 309:657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of those amino acids are substituted with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0144] "Framework Regions" (FRs) are the variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH, and FR H4 corresponds to FR 4 of VH. Similarly, the FRs of VL are also referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to FR 4 of VL.

[0145] As used herein, the term "Fv" shall be taken to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which the VL and VH combine to form a complex with an antigen-binding domain, i.e., capable of specifically binding to an antigen. The VH and VL forming the antigen-binding domain may be a single polypeptide chain or different polypeptide chains. Furthermore, the Fv of the invention (and any protein of the invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. The term shall be understood to encompass fragments directly derived from antibodies produced using recombinant means, proteins corresponding to such fragments. In some instances, the VH is not linked to the heavy chain constant domain (CH)1 and / or the VL is not linked to the light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher complexes or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain to obtain a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to obtain a molecule consisting of an intact light chain and a portion of the heavy chain containing the VH and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked together using, for example, a leucine zipper or a CH3 domain.A "single-chain Fv" or "scFv" is a recombinant molecule comprising an antibody variable region fragment (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0146] As used herein, the term "bind" in reference to the interaction of an antigen-binding site or its antigen-binding domain with an antigen means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures, rather than proteins in general. If an antibody binds to epitope "A", then in a reaction involving labeled "A" and a protein, the presence of a molecule containing epitope "A" (or free unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0147] As used herein, the terms "specifically binds to" or "specifically binds to" shall be taken to mean that the antigen binding sites of the invention react or bind to a particular antigen or cell expressing it more frequently, more rapidly, for longer duration and / or with higher affinity as compared to alternative antigens or cells. For example, the antigen binding site binds to PSMA with substantially higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than it does to other antigens.

[0148] As used herein, the term "epitope" (synonym "antigenic determinant") shall be understood to mean the region of a cell surface protein (such as PSMA) to which an antigen binding site, including the antigen binding domain of an antibody, binds.

[0149] As used herein, the term "condition" refers to a disruption or interruption of normal function and should not be limited to any particular condition, but includes a disease or disorder.

[0150] As used herein, "preventing", "prevent" or "prevention" includes arresting or hindering the onset of at least one symptom of a condition by administering an antigen binding site of the invention. The term also encompasses treatment of a subject in remission to prevent or hinder recurrence.

[0151] As used herein, the terms "treating," "treat" or "treatment" include reducing or eliminating at least one symptom of a particular disease or condition by administering an antigen binding site described herein.

[0152] As used herein, the term "subject" shall be taken to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0153] Engineered antibodies The present invention relates, in part, to modifications to IgG antibodies that comprise one or more amino acid substitutions to regions of the antibody that reduce or eliminate the affinity of the antibody for FcRn, thereby decreasing the serum half-life of the antibody.

[0154] It will be appreciated that, in accordance with the present invention, any antibody for which a reduced serum half-life is desired may be modified according to the methods described herein. It will further be appreciated that, in preferred embodiments, antibodies modified to have a reduced serum half-life are antibodies that are useful for diagnostic or therapeutic applications, more particularly theranostic applications.

[0155] Examples of antibodies suitable for use with the methods of the present invention include trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab (Avastin®), dinutuximab (Unituxin®), pancreatic leukemia antibody (PLA ... Examples of antibodies that may be used include tumumab (Vectibix®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), tositumomab (Bexxar®), and ibritumomab (Zevalin®). However, it will be understood that the present invention is not limited to a particular antibody, provided that the antibody is otherwise susceptible to binding by FcRn.

[0156] The present invention also contemplates the use of antibody drug conjugates that target tumor antigens, where the conjugate comprises a cytotoxic payload. Examples of such antibodies include gemtuzumab (Mylotarg®), brentuximab (Adcetris®), inotuzumab (Besponsa®), glenbatumumab (CDX-011), anetumab (BAY 94-9343), mirvetuximab (IMGN853), depatuxizumab (ABT-414), rovalpituzumab (Rova-T) and rovalpituzumabutarilin (SGN-CD33A). Further examples include Lambert et al., 2017, Adv Ther (2017) 34:1015-1035, which is incorporated herein by reference.

[0157] In certain embodiments, antibodies suitable for modification according to the present invention to reduce affinity for FcRn are those having one or more of the sequences shown in Table 1.

[0158] The invention also provides antigen binding sites or nucleic acids encoding same that have at least 80% identity to the sequences disclosed herein, hi one example, an antigen binding site or nucleic acid of the invention comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0159] Alternatively or additionally, the antigen binding site may be any of the V H Or V LThe CDRs of the present invention may comprise at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to the CDRs of the present invention (e.g., three CDRs).

[0160] In another example, a nucleic acid of the invention comprises a sequence that is at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence encoding an antigen binding site having a function as described herein according to any of the examples. The invention also encompasses nucleic acids encoding antigen binding sites of the invention that differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0161] The percent identity of a nucleic acid or polypeptide is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.

[0162] The present invention also contemplates nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding antigen-binding sites as described herein. "Moderate stringency" is defined herein as hybridization and / or washing in 2xSSC buffer, 0.1% (w / v) SDS at a temperature in the range of 45°C to 65°C or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing in 0.1xSSC buffer, 0.1% (w / v) SDS or lower salt concentration at a temperature of at least 65°C or equivalent conditions. References herein to a particular level of stringency include hybridization and / or washing in a buffer other than SSC known to those of skill in the art. / equivalent conditions using hybridization solutions. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as melting temperature or Tm) are known in the art. A temperature similar (e.g., within 5°C or within 10°C) or equal to the Tm of the nucleic acid is considered high stringency. Intermediate stringency is considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.

[0163] The present invention also contemplates mutant forms of the antigen binding sites of the present invention that contain one or more conservative amino acid substitutions compared to the sequences shown herein. In some examples, the antigen binding site contains up to 10, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathy and / or hydrophilicity.

[0164] Families of amino acid residues having similar side chains have been defined in the art and include 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indexes are described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982, and hydrophilicity indexes are described, for example, in U.S. Pat. No. 4,554,101.

[0165] The present invention also contemplates non-conservative amino acid changes. For example, substitutions of charged amino acids with other charged amino acids and neutral or positively charged amino acids are of particular interest. In some examples, the antigen-binding site contains 10 or less, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0166] In one example, the mutations occur within the FRs of the antigen binding domain of the antigen binding site of the invention. In another example, the mutations occur within the CDRs of the antigen binding site of the invention.

[0167] Exemplary methods for generating mutant forms of an antigen-binding site include: Mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO 1999 / 058661); introducing a nucleic acid encoding the polypeptide into a mutagen cell, such as XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cell (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, Nature 370:389-91, 1994; and ·For example, Dieffenbach (ed) and Dveksler (ed) (PCR Primer: A Laboratory Manual, Cold Spring These include site-directed mutagenesis as described in (Harbor Laboratories, NY, 1995).

[0168] Exemplary methods for determining the biological activity (e.g., antigen binding) of a mutant antigen binding site of the invention will be apparent to one of skill in the art and / or are described herein. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein. It is stated in the book.

[0169] Constant region The present invention encompasses antigen-binding sites and / or antibodies described herein that comprise an antibody constant region, including antigen-binding fragments of an antibody fused to Fc.

[0170] The sequences of constant regions useful for generating the proteins of the invention can be obtained from a number of different sources. In some examples, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is a human isotype IgG4 or stabilized IgG4 constant region.

[0171] Preferred modifications The present invention specifically contemplates modifications to antibodies or antigen binding sites, including the Fc region or constant region.

[0172] The neonatal Fc-receptor (FcRn) is important for the in vivo metabolic fate of IgG class antibodies. FcRn functions to rescue IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein consisting of two polypeptides: the 50 kDa class I major histocompatibility complex-like protein (a-FcRn) and the 15 kDa p2-microglobulin (β2ηι). FcRn binds with high affinity to the CH2-CH3 portion of the Fc-region of antibodies of the IgG class. The interaction between antibodies of the IgG class and FcRn is pH-dependent and occurs with a 1:2 stoichiometry, i.e., one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc-region polypeptides (see, e.g., Huber, AH, et al, J. Mol. Biol. 230 (1993) 1077-1083).

[0173] Thus, the in vitro FcRn binding properties / characteristics of IgG are indicative of its in vivo pharmacokinetic properties in the blood circulation. The interaction between FcRn and the Fc-region of IgG class antibodies involves distinct amino acid residues in the heavy chain CH2- and CH3-domains.

[0174] Various mutations that affect FcRn binding and thereby affect half-life in blood circulation are known. Fc-region residues important for mouse Fc-region-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues Ile253, His310, His433, Asn434 and His435 (numbered according to the EU index numbering system) are involved in the interaction (Medesan, C, et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542-548). (Using the Kabat system, the relevant residues are Ile266, His329, His464, Asn465 and His466). Residues Ile253, His310 and His435 were found to be important for the interaction of the human Fc-region with murine FcRn (Kim, JK, et al, Eur. J. Immunol. 29 (1999) 2819-2885).

[0175] More specifically, the antibody may contain one or more amino acid substitutions that reduce the half-life of the protein. For example, the antibody contains an Fc region that contains one or more amino acid substitutions that reduce the affinity of the Fc region for neonatal Fc region (FcRn).

[0176] The present invention also provides an antibody having a constant region substantially identical to a naturally occurring class IgG antibody constant region, wherein at least one amino acid residue selected from the group consisting of residues His310, His435 and Ile253 differs from that present in a naturally occurring class IgG antibody, thereby altering the FcRn binding affinity and / or serum half-life of said antibody relative to the naturally occurring antibody. In a preferred embodiment, the naturally occurring class IgG antibody comprises the heavy chain constant region of a human IgG1, IgG2, IgG2M3, IgG3 or IgG4 molecule.

[0177] Also in a preferred embodiment, amino acid residue 310 or residue 435 from the heavy chain constant region of an antibody having substantially the same constant region as a naturally occurring class IgG antibody is any amino acid other than histidine that reduces the affinity of the constant region for FcRn. For example, the amino acid at residue 310 or 435 can be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0178] or amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine or alanine. In another preferred embodiment, the antibody having a constant region substantially identical to a naturally occurring class IgG antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0179] In a preferred embodiment of the invention, the binding affinity to FcRn and / or serum half-life of the modified antibody is reduced by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold. In a preferred embodiment of the invention, the binding affinity to FcRn and / or serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

[0180] In addition, the antibodies of the invention may contain one or more mutations that alter the affinity of the antibody for any one or more Fc gamma receptors.

[0181] In a preferred embodiment of the present invention, the Fc region of the constant region retains the ability to induce effector function. In one example, the Fc region of the constant region comprises one or more amino acid substitutions that modulate effector function, including increasing effector function compared to wild-type IgG.

[0182] In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region containing protein are known in the art and / or described herein.

[0183] In one example, the amino acid substitution that alters the ability of the antibody to induce an effector function is an amino acid substitution at residue Ile253 from the heavy chain constant region. In one example, the substitution is any selected from alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine. to an amino acid which reduces the ability of the antibody to induce effector function. In a preferred embodiment, the substitution from He at residue 253 is to arginine, proline or aspartate, more preferably to alanine.

[0184] In one example, the Fc region is an IgG4 Fc region (i.e. from an IgG4 constant region), such as a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to those of skill in the art and / or available in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0185] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" is understood to mean an IgG4 constant region that has been modified to reduce its tendency to undergo Fab arm exchange or to form half antibodies or to form half antibodies. "Fab arm exchange" refers to a type of protein modification for human IgG4 in which an IgG4 heavy chain and associated light chain (half molecule) are exchanged with a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can also be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. "Half antibodies" are formed when an IgG4 antibody dissociates to form two molecules, each of which contains a single heavy chain and a single light chain.

[0186] In one example, a stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system. In human IgG4, this residue is generally a serine. After substitution of the proline with serine, the IgG4 hinge region contains the sequence CPPC. In this respect, the skilled artisan will recognize that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, conferring flexibility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in the inter-heavy chain disulfide bond. It is generally defined as the stretch from Glu226 to Pro243 of human IgG1 according to the Kabat numbering system (or from Glu216 to Pro230 using the EU index). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine ​​residues to form inter-heavy chain disulfide (SS) bonds at the same positions (see, for example, WO 2010 / 080538).

[0187] A further example of a stabilized IgG4 antibody is an antibody in which the arginine at position 409 (according to the EU numbering system) of the heavy chain constant region of human IgG4 is replaced with lysine, threonine, methionine or leucine (e.g. as described in WO 2006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of alanine, valine, glycine, isoleucine and leucine at the position corresponding to: 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e. the CPPC sequence) (as described above).

[0188] In another example, the Fc region is a region that has been modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region that contains substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region contains the following changes E233P, L234V, L235A, and and G236 deletions and / or one or more of the following changes A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Further examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138 2006; and / or Hezareh J Virol;75:12161-12168,2001).

[0189] In another example, the Fc region can comprise at least one C region, e.g., from an IgG4 antibody. H 2 domain and at least one C from an IgG1 antibody H and 3 domains, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO 2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S and 427F.

[0190] antibody generation Preferably, the antigen binding sites described herein, by any of the examples, are recombinant.

[0191] In the case of recombinant proteins, the nucleic acid encoding same can be cloned into an expression construct or vector, which is then transfected into a host cell, such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a myeloma cell (which does not otherwise produce the protein). Exemplary cells used for expression of proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and are described, for example, in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, U.S. Pat. No. 4,816,567 or U.S. Pat. No. 5,530,101.

[0192] Once isolated, the nucleic acid is inserted into an expression construct or expression vector so as to be operably linked to a promoter for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0193] As used herein, the term "promoter" should be interpreted in its broadest context and includes the transcriptional regulatory sequences of genomic genes, including the TATA box or initiator elements required for correct transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of the nucleic acid in response to, for example, developmental and / or external stimuli or in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe recombinant, synthetic or fusion nucleic acids or derivatives that confer, activate or enhance expression of a nucleic acid to which they are operably linked. Exemplary promoters include those that are capable of expressing a nucleic acid that is ... Alternatively, the nucleic acid may include additional copies thereof to further enhance expression and / or alter spatial and / or temporal expression of the nucleic acid.

[0194] As used herein, the term "operably linked" refers to the positioning of a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0195] Numerous vectors are available for expression in cells. Vector components generally include one or more of the following, but are not limited to: signal sequence, protein coding sequence (e.g., from the information provided herein), enhancer element, promoter, and transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0196] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; hybrid regulatory elements comprising the CMV enhancer / β-actin promoter or immunoglobulin promoters or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0197] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0198] Means for introducing isolated nucleic acids or expression constructs containing same into cells for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran mediated transfection, liposome-mediated transfection, for example by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and biolistics, for example by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA), among others.

[0199] Host cells used for protein production may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for the culture of the other cell types discussed herein are known in the art.

[0200] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0201] If the antigen-binding site is secreted into the culture medium, the supernatant from such an expression system may first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants. Alternatively or additionally, the supernatant may be filtered and / or separated from the cells expressing the protein, for example using continuous centrifugation.

[0202] Antigen binding sites prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example, in WO 99 / 57134 or in Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0203] Those skilled in the art will also recognize that proteins can be modified to include tags, such as poly-histidine tags, such as hexa-histidine tags, or influenza virus hemagglutinin (HA) tags, or simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags, to facilitate purification or detection. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins that include hexa-histidine tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-histidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0204] Binding of radioisotopes to antibodies In any of the embodiments of the invention, the antibodies described herein may be directly or indirectly conjugated to a diagnostic or therapeutic agent, preferably the diagnostic or therapeutic agent is a radioisotope.

[0205] An example of a suitable isotope is actinium-225 ( 225 Ac), astatine-211( 211At), Bismuth-212 and Bismuth-213 ( 212 Bi, 213 Bi), copper-64 and copper-67 ( 64 Cu, 67 Cu), Gallium-67 and Gallium-68 ( 67 Ga and 68 Ga), Indium-111 ( 111 In), iodine-123, -124, -125 or -131 ( 123 I, 124 I, 125 I, 131 I)( 123 I), lead-212( 212 Pb), Lutetium-177( 177 Lu), Radium-223 ( 223 Ra), Samarium-153( 153 Sm), Scandium-44 and Scandium-47 ( 44 Sc, 47 Sc), Strontium-90 ( 90 Sr), Technetium-99 ( 99m Tc), yttrium-86 and yttrium-90 ( 86 Y, 90 Y), Zirconium-89( 89 Those skilled in the art will know which radioisotopes are preferred for use as diagnostic agents and which are preferred for use as therapeutic agents. cormorant.

[0206] It will be appreciated that radioisotopes may be conjugated to the antibodies of the invention directly (via a chelating agent or prosthetic group or linker) or indirectly via attachment to a single or multiple amino acid residues in the antibody (e.g., halogenation of tyrosine residues).

[0207] In alternative embodiments, chelators or linkers may be used to conjugate the radioisotope to the antibody. In one example, the antibody may be conjugated to a chelating moiety selected from the group consisting of TMT (6,6″-bis[N,N′”,N′″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-N-N′,N″(N′″-tetraacetic acid, also known as tetraxetane), TCMC (tetra-primary amide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane- triacetic acid), Diamsar (3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), DTPA (pentetic acid or diethylenetriaminepentaacetic acid), CHX-A''-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1 ,4,8,11-tetraazacyclotetradecane-1,4,8), 11-tetraacetic acid, Te2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED, DFO (deferoxamine), DFOsq (DFO-squaramide), and HOPO (3,4,3-(LI-1,2-HOPO) or other chelating agents described herein.

[0208] Chelators bearing radiometals and other halogenated radioisotopes can be attached to the antibodies of the invention via one or more amino acid residues or reactive moieties in the antibody, including, but not limited to, one or more lysine residues, tyrosine residues, or thiol moieties.

[0209] In another example, the modified antibody is conjugated to a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or using any amine or thiol modification chemistry known in the art.

[0210] Those skilled in the art will be familiar with standard methods for conjugating chelators to antibodies and their derivatives or fragments. In addition, those skilled in the art will be familiar with approaches for selecting relevant chelators to pair with radiometals, as described, for example, in Chem. Soc. Rev., 2014, 43, 260 (incorporated herein by reference).

[0211] Assay of antigen-binding site activity Binding to PSMA From the disclosure herein, it will be apparent to one skilled in the art that the preferred antigen-binding sites of the present invention bind to PSMA. Methods for assessing protein binding are known in the art and include, for example, Scopes (Protein purification: principles of Such methods generally involve immobilizing an antigen-binding site and contacting it with labeled antigen. After washing to remove non-specifically bound proteins, the label and, consequently, the amount of bound antibody are detected. However, the antigen binding site may be labeled and the antigen immobilized. Panning type assays may also be used. Alternatively or additionally, surface plasmon resonance assays may be used.

[0212] Therapeutic, Diagnostic and Theranostic Methods The antibodies of the present invention are useful in the treatment of a number of conditions which require treatment by radioimmunotherapy. Typically such conditions include cancer.

[0213] Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors, including tumors in the anal tissue, bile duct, bladder, blood cells, intestine, brain, breast, carcinoid, cervix, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph node, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin (e.g., squamous cell carcinoma), sarcoma, stomach, testis, thyroid, vagina, vulva. Soft tissue tumors include benign neurilemmoma monosomy, desmoid tumor, lipoblastoma, lipoma, uterine fibroids, clear cell sarcoma, dermatofibrosarcoma, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, liposarcoma myxoid type, alveolar rhabdomyosarcoma, and synovial sarcoma. Specific bone tumors include nonossifying fibroma, solitary bone cyst, enchondroma, aneurysmal bone cyst, osteoblastoma, chondroblastoma, chondromyxoid fibroma, osteogenic fibroma and adamantinoma, giant cell tumor, fibrous dysplasia, Ewing's sarcoma, eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma and metastatic carcinoma. Leukemias include acute lymphocytic, acute myeloblastic, chronic lymphocytic and chronic myelogenous.

[0214] Other examples include breast tumors, colorectal tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, hepatocellular / bile duct tumors, carcinoma, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, non-seminomatous epithelioma, stromal Leydig cell tumor, Sertoli cell tumor, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral cavity tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, transvaginal tumors, and Wilms' tumor.

[0215] Preferably, the antigen binding sites of the invention are useful for treating cancers characterised by the presence of PSMA, for example antibodies that bind PSMA are useful for treating cancers characterised by increased expression of PSMA, including prostate cancer.

[0216] One of skill in the art would be familiar with how to select appropriate diagnostic agents for use with the antibodies of the present invention, including radioisotopes used in radioimaging to diagnose the conditions disclosed herein. Moreover, one of skill in the art would be familiar with the imaging techniques for use with the diagnostic reagents described herein.

[0217] As used herein, theranostic methods are methods of in vitro and / or in vivo visualization, identification and / or detection of tumor cells and / or metastases as well as methods of treating cancer.

[0218] In one embodiment, the present invention provides (1) administering to a patient or subject a diagnostically effective amount of an antibody of the invention, where the antibody contains at least one diagnostically useful label; (2) administering a therapeutically effective amount of an antibody of the present invention to a patient or subject in need thereof, the antibody comprising a tumor therapeutic agent (e.g., a radioisotope, a toxin, a drug); Theranostic methods include:

[0219] Preferably, steps 1 and 2 are performed consecutively, and the antibodies in steps 1 and 2 are the same. be.

[0220] Antibody-binding domain-containing proteins Single Domain Antibodies In some examples, an antigen binding site or protein of the invention is or comprises a single domain antibody (which is used interchangeably with the term "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or a portion of the heavy chain variable region of an antibody. In particular examples, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516).

[0221] Diabodies, triabodies, tetrabodies In some examples, a protein of the invention is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in, for example, WO 98 / 044001 and / or WO 94 / 007921.

[0222] For example, a diabody is a protein that includes two binding polypeptide chains, each of which has the structure V L -XV H Or V H -XV L Including V L is the antibody light chain variable region, V H is an antibody heavy chain variable region, and X is a V H and V L a linker that contains insufficient residues to allow the V of one polypeptide chain to bind (or form an Fv) or is absent H is the V of the other polypeptide chain. L to form an antigen-binding domain, i.e., an Fv molecule capable of specifically binding to one or more antigens. L and V H may be the same in each polypeptide chain, or V L and V H can be different in each polypeptide chain to form a bispecific diabody (i.e., containing two Fvs with different specificities).

[0223] Single chain Fv (scFv) Those skilled in the art will appreciate that scFvs are V H and V L Includes the area, V H and V L and a polypeptide linker between the scFv and the V H and V Lwill recognize that the linker comprises more than 12 amino acid residues and (Gly4Ser)3 is one of the more preferred linkers for scFv.

[0224] The present invention also contemplates disulfide-stabilized Fv (i.e., diFv or dsFv), in which a single cysteine ​​residue is located at the V H FR and V L and cysteine ​​residues are linked by disulfide bonds to provide a stable Fv.

[0225] Alternatively or additionally, the invention encompasses dimeric scFvs, i.e., a protein comprising two scFv molecules that are non-covalently or covalently linked, for example, by a leucine zipper domain (e.g., from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow both scFv formation and binding to antigen, for example, as described in US Patent Publication No. 20060263367.

[0226] Heavy Chain Antibodies Heavy chain antibodies are structurally distinct from many other forms of antibodies in that they contain heavy chains but no light chains. Thus, these antibodies are also called "heavy chain-only antibodies." Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNAR).

[0227] The variable regions present in naturally occurring heavy chain antibodies are generally similar to the heavy chain variable regions present in conventional four-chain antibodies ("V H The light chain variable region (called the "V domain") present in conventional four-chain antibodies L domains) in camelid antibodies. HH In IgNARs, these are referred to as "domains" and in IgNARs, V-NARs.

[0228] General descriptions of heavy chain antibodies and variable regions thereof from camelids and methods of their production and / or isolation and / or use can be found, inter alia, in the following references: WO 94 / 04678, WO 97 / 49805 and WO 97 / 49805.

[0229] A general description of heavy chain antibodies and their variable regions from cartilaginous fish and methods of producing and / or isolating and / or using them can be found, inter alia, in WO 2005 / 118629.

[0230] Other antibodies and proteins containing their antigen-binding domains The present invention contemplates the following other antibodies and proteins comprising antigen-binding domains thereof: (i) "Key and hole" bispecific proteins, such as those described in U.S. Pat. No. 5,731,168; (ii) heteroconjugate proteins, such as those described in U.S. Pat. No. 4,676,980; (iii) heteroconjugate proteins produced using chemical cross-linkers, such as those described in U.S. Pat. No. 4,676,980; and (iv) Fab3 (e.g., as described in EP 19930302894).

[0231] composition In some examples, the antigen binding sites described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intracerebroventricularly, via an implanted reservoir, or in any other convenient form in dosage formulations containing conventional non-toxic pharma- ceutically acceptable carriers. The term "parenterally" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, intrasternal and intracranial injection or infusion techniques.

[0232] Methods for preparing antigen-binding sites into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0233] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, e.g., intravenous administration or administration into a body cavity or into a lumen or joint of an organ. A composition for administration will usually comprise a solution of the antigen-binding site dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, e.g., saline, etc., can be used. The compositions can include pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding site of the present invention in these formulations can vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, can also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0234] Dosage and timing of administration Appropriate dosages of the antigen-binding sites of the present invention will vary depending on the specificity of the antigen-binding site, the condition being treated and / or the subject being treated. It is within the ability of a skilled physician to determine appropriate dosages, for example, by starting with a suboptimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies are used to determine appropriate dosages for treatment / prophylaxis, where an appropriate dose is determined to be greater than or equal to the ED of the active compound with little or no toxicity. 50 The therapeutically / prophylactically effective dose can be estimated initially by cell culture assays. Dosages are within a circulating concentration range that includes the IC 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range that includes a compound concentration or amount that achieves a half-maximal inhibition of symptoms (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0235] In some examples, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0236] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces one or more symptoms of a clinical condition described herein to a level below that observed and accepted as a clinical diagnosis or clinical feature of the condition. The amount administered to a subject will depend on the specific characteristics of the condition being treated, the type and stage of the condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and weight. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. Thus, the term should not be construed to limit the invention to a specific amount, such as weight or amount of protein, and the invention encompasses any amount of antigen-binding site sufficient to achieve the stated result in a subject.

[0237] As used herein, the term "prophylactically effective amount" shall be taken to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One of skill in the art will recognize that such amounts will vary depending, for example, on the particular antigen-binding site administered and / or the particular subject and / or the type or severity or level of the condition and / or predisposition (genetic or otherwise) to the condition. Thus, this term should not be construed to limit the invention to a particular amount, for example, weight or amount of antigen-binding site, but rather the invention encompasses any amount of antigen-binding site sufficient to achieve a specified result in a subject.

[0238] kit The invention additionally includes kits comprising one or more of the following: (i) an antibody of the invention or an expression construct encoding same; (ii) a molecule of the invention; (iii) a conjugate of the present invention; or (iii) A pharmaceutical composition of the present invention.

[0239] In the case of a kit for the detection of cancer, the kit may further comprise a detection means, for example bound to the antigen-binding site of the present invention.

[0240] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharma- ceutically acceptable carrier.

[0241] Optionally, the kits of the invention are packaged with instructions for use in any of the examples of the methods described herein.

[0242] [Table 1]

[0243] [Table 2]

[0244] [Table 3]

[0245] [Table 4]

[0246] [Table 5]

[0247] [Table 6] EXAMPLES

[0248] Example 1: Antibodies for binding to PSMA overview The antibody VH gene sequences of the two antibodies, ANT4044 and ANT4044-A2, were cloned into three different human IgG dual expression vectors encoding an unmodified IgG1, an IgG1 with mutations H310A and H435Q (which abolish FcRn binding and protein A binding (Andersen, et al., 2012)) (referred to as IgG1(H310A, H435Q)), and a modified IgG4 with the same FcRn abolishing mutations as above, along with a hinge-stabilizing S228P mutation (Angal, et al., 1993) and an Fc-silencing L235E mutation (Reddy, et al., 2000) (referred to as IgG4(S228P, L235E, H310A, H435Q)). Each dual expression vector also contained an antibody Vκ gene sequence common to both ANT4044 and ANT4044-A2.

[0249] A total of five antibodies were transiently transfected and expressed in CHO cells and purified using either Protein A (ANT4044-A2 IgG1) or Protein G (both ANT4044 and ANT4044-A2 as both IgG1 (H310A, H435Q) and IgG4 (S228P, L235E, H310A, H435Q)). Affinity chromatography was followed by preparative size exclusion chromatography (SEC).

[0250] Antibody integrity was assessed by SDS-PAGE, analytical SEC, thermal stability and antigen binding to PSMA by Biacore. Further testing was performed against a panel of human Fc gamma receptors (FcγRlllA176F, FcγRlllA176V, FcγRlllB, FcγRlIA167R, FcγRllA167H, FcγRllB, FcgRl) and the neonatal receptor, FcRn, using Biacore single cycle analysis.

[0251] Methods and Results Construction of antibody-expression plasmids The VH and Vκ sequences of humanized antibody ANT4044 and affinity matured humanized antibody ANT4044-A2 were used to generate DNA fragments with flanking restriction enzyme sites for cloning into the pANT dual expression vectors IgG1 (pANT18), IgG1 (H310A, H435Q) (pANT71) and IgG4 (S228P, L235E, H310A, H435Q) (pANT73). The VH region was cloned between the Mlu I and Hind III restriction sites and the Vκ region was cloned between the BssH II and BamH I restriction sites in each isotype vector. All five constructs were verified by DNA sequencing.

[0252] Transient expression of antibodies Endodoxin-free DNA corresponding to the five antibody constructs was prepared and transiently transfected into CHO-S cells (ThermoFisher, Loughborough, UK) using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA). Following harvest, cells were cultured at 3x10 in CD OptiCHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-glutamine (ThermoFisher, Loughborough, UK) and 1x hypoxanthine-thymidine (ThermoFisher, Loughborough, UK). 6 Twenty-four hours after transfection, the culture temperature was reduced to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added.

[0253] Cultures were fed daily with the addition of 3.6% (of starting volume) feed (2.5% CHO CD Efficient Feed A (ThermoFisher, Loughborough, UK), 0.5% Yeastolate (BD Biosciences, Oxford, UK), 0.25 mM Glutamax (ThermoFisher, Loughborough, UK) and 2 g / L glucose (Sigma, Dorset, UK)). IgG supernatant titers were monitored by IgG ELISA and transfected cells were cultured for up to 14 days before supernatants were harvested.

[0254] Antibody purification Cell culture supernatants were passed over either Protein A (ANT4044-A2 IgG1) or Protein G (both ANT4044 and ANT4044-A2 as both IgG1 (H310A, H435Q) and IgG4 (S228P, L235E, H310A, H435Q)) Sepharose columns (GE Healthcare, Little Chalfont, UK). All antibodies were buffer exchanged into 1x PBS, pH 7.2. Protein A or Protein G purified material was purified using a HiLoad™ 26 / 600 Superdex™ 200 pg preparative SEC column (GE Pharma) using 1x PBS as the mobile phase. The antibodies were run on a 50-mL ELISA kit (Healthcare, Little Chalfont, UK) while monomeric fractions were collected, pooled and filter sterilized. SEC profiles revealed that the antibodies, particularly ANT4044-A2, when expressed as IgG4 (S228P, L235E, H310A, H435Q), showed higher levels of aggregation (up to 22%) compared to antibodies expressed as other IgG isotypes.

[0255] Antibodies were quantified by measuring OD280nm and using the extinction coefficient (Ec(0.1%)) based on their predicted amino acid sequence.

[0256] Analytical SEC and SDS-PAGE Stock ANT4044 IgG1 and Protein A or Protein G followed by preparative SEC purified material were analyzed by analytical SEC using a Superdex™ 200 Increase 10 / 300 GL analytical column (GE Healthcare, Little Chalfont, UK) and 1xPBS as the mobile phase. The elution profile was typical of a correctly folded monomeric species of IgG. The antibodies were also analyzed by non-reducing and reducing SDS-PAGE. Bands corresponding to the expected sizes of VH and Vκ chains were observed.

[0257] Thermal stability analysis To assess the thermal stability of the five purified antibodies from stocks and ANT4044 IgG1, the melting temperature (temperature at which 50% of the protein domains are unfolded) was determined using a fluorescence-based thermal shift assay. All antibodies were diluted to a working concentration of 100 μg / ml in 1×PBS containing SYPRO® Orange (ThermoFisher, Loughborough, UK) and subjected to a temperature gradient from 25° C. to 99° C. over 56 min on a StepOnePlus real-time PCR system (ThermoFisher, Loughborough, UK). Melting curves were analyzed using Protein Thermal Stability Software (version 1.2) and Tm was calculated based on the first derivative data.

[0258] For both ANT4044 and ANT4044-A2, the IgG1 backbone appeared to be the most thermally stable, with the lowest melting temperature for both being 69.3° C. Comparing the different IgG backbones, ANT4044 showed greater thermal stability compared to ANT4044-A2.

[0259] Assessment of binding to PSMA Multi-cycle kinetic analysis was performed on each of the purified antibodies to evaluate binding to prostate-specific membrane antigen (PSMA). Analysis was performed using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V3.0.1 (Uppsala, Sweden). For direct comparison, all antibodies were captured on a Protein G chip (GE Healthcare, Uppsala, Sweden).

[0260] Purified antibodies were diluted to a concentration of 1 μg / ml in HBS-EP+. At the start of each cycle, each antibody was captured onto the Protein G surface to obtain a RL of approximately 50 RU. After capture, the surface was allowed to stabilize. Kinetic data were acquired using a flow rate of 35 μl / min to minimize any potential mass transfer effects. For kinetic analysis, PSMA (R&D Systems, Minneapolis, USA) was used. Multiple replicates of a blank (PSMA) and replicates of a single concentration of analyte were programmed into the kinetic run to check the stability of both the surface and analyte over the kinetic cycles. For kinetic analysis, a 2-fold dilution range was used from 25 nM The concentration of PSMA was chosen to be ~1.5625 nM. The association phase of PSMA was monitored for 600 s and the dissociation phase was monitored for 2400 s. Regeneration of the Protein G surface was performed at the end of each cycle using two injections of 10 mM glycine-HCL (pH 1.5) containing 0.5% P20.

[0261] The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface and the overall Rmax parameter was used in the one-to-one binding model. Relative KD was calculated by dividing the KD of each antibody by the KD of ANT4044 IgG1 on the same chip.

[0262] Assessment of binding to human FcRn Binding of purified antibodies to FcRn was assessed by steady-state affinity analysis using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V3.0.1 (Uppsala, Sweden). FcRn (Sino Biological, Beijing, China) was coated onto a CM5 chip at 10 μg / mL in sodium acetate (pH 5.5) using standard amine coupling to 300 RU.

[0263] Purified antibodies were titrated in 5-point dilutions from 37 nM to 3000 nM in PBS containing 0.05% P20 at either pH 6.0 or pH 7.4. Antibodies were passed over the chip at increasing concentrations at a flow rate of 30 μl / min and 25° C. Injection time was 30 s and dissociation time was 100 s. Following a single dissociation, the chip was regenerated with 0.1 M Tris pH 8.0.

[0264] As expected, the H310A H435Q mutation significantly reduced antibody binding to FcRn at pH 6.0. Both the ANT4044 and ANT4044-A2 antibodies tested as unmodified IgG1s showed similar affinity for FcRn at pH 6.0. Little binding was observed at pH 7.4 for either antibody.

[0265] Assessment of binding to human Fc gamma receptors Binding of purified antibodies to high and low affinity Fc gamma receptors was assessed by single cycle analysis using a Biacore T200 (serial no. 1909913) instrument running Biacore T200 evaluation software V3.0.1 (Uppsala, Sweden) operated at a flow rate of 30 μl / min. Human Fc receptors, FcγRI, FcγRIIa (both 167R and 167H polymorphisms), FcγRIIb, FcγRIIIa (both 176F and 176V polymorphisms) and FcγRIIIb, were obtained from Sino Biological (Beijing, China). FcγRs were captured using Hiscapture kits (GE Healthcare, Little Chalfont, UK) onto CM5 sensor chips pre-coupled using standard amine chemistry.

[0266] At the start of each cycle, His-tagged Fcγ receptors diluted in HBS-P+ were loaded to a specific RU level. A five-point, 3-fold dilution range of antibody with no regeneration between concentrations was used for each receptor. In all cases, after dissociation, the chip was regenerated with two injections of glycine (pH 1.5). The signal from the reference channel Fc1 (blank) was subtracted from the signal of the receptor-loaded Fc to correct for differences in non-specific binding to the reference surface.

[0267] The sensorgrams were analyzed for 1:1 kinetics for the high affinity Fc gamma receptor FcγRI and by steady state binding for the low affinity Fc gamma receptor.

[0268] Representative sensorgram ANT4044 and ANT4044-A2 expressed as unmodified IgG1 bound to all high and low affinity human activating Fcγ receptors. Introduction of the H310A and H435Q mutations in IgG1 did not affect this binding, but a slight trend towards reduced binding to low affinity human Fcγ receptors was observed. As expected, the IgG4(S228P, L235E, H310A, H435Q) antibody showed significantly reduced binding to all activating Fcγ receptors. All antibodies tested showed low affinity binding to the inhibitory receptor FcγRIIB. Thus, both IgG1 and IgG1(H310A, H435Q) may be able to stimulate effector functions, whereas IgG4(S228P, L235E, H310A, H435Q) is less likely to stimulate effector functions.

[0269] conclusion Variable heavy and light chain sequences corresponding to the humanized anti-PSMA antibody ANT4044 and its affinity matured variant ANT4044-A2 were cloned into dual expression vectors encoding either unmodified human IgG1, human IgG1 with mutations H310A and H435Q (to abolish FcRn binding) or human IgG4 (S228P, L235E, H310A, H435Q). CHO cells were transiently transfected and the five antibodies were purified by protein A or protein G affinity chromatography and preparative SEC. Analytical SEC revealed a profile consistent with monomeric IgG with little evidence of aggregation. All antibodies, including ANT4044 IgG1 (from stock), were characterized in terms of their thermal stability and binding to PSMA, human FcRn and human Fcγ receptors.

[0270] For both ANT4044 and ANT4044-A2, the IgG1 backbone appeared to be the most thermally stable, but the ANT4044 antibody showed greater thermal stability compared to the ANT4044-A2 antibody. All antibodies in both IgG1 (H310A, H435Q) and IgG4 (S228P, L235E, H310A, H435Q) formats showed binding to PSMA similar to their counterparts expressed as unmodified IgG1. The affinity matured ANT4044-A2 antibody showed 2-3 fold greater affinity for PSMA compared to ANT4044.

[0271] The binding to FcRn at pH 6.0 was significantly reduced by the introduction of the H310A and H435Q mutations, whereas the binding to FcRn at pH 7.4 was hardly observed for any of the antibodies.

[0272] Analysis of antibody binding to human Fcγ receptors confirmed that the L235E mutation (S228P, L235E, H310A, H435Q) in the antibody expressed as IgG4 abolished binding. Mutations H310A and H435Q did not significantly affect antibody binding to human Fcγ receptors, and therefore both IgG1 and IgG1(H310A, H435Q) are expected to exhibit similar effector function properties.

[0273] Example 2: Antibody Conjugation The antibodies ANT4044-IgG1, ANT4044-A2-IgG1, ANT4044-IgG1 H310A H435Q (also known as ANT4044-IgG1-2M) and ANT4044-IgG4 S228P L235E H310A H435Q (also known as ANT4044-IgG4-4M) were conjugated to either ThioBridge™-PEG(6u)-DOTA reagent or NHS-DOTA reagent.

[0274] ThioBridge™ is a proprietary disulfide conjugation linker from PolyTherics and has been described.

[0275] DOTA is a chelator payload, 1,4,7,10-tetraazacyclododecane- It is 1,4,7,10-tetraacetic acid monoamide.

[0276] ThioBridge™ DOTA conjugation evaluation: ANT4044-IgG1 was prepared as a 6-10 mg / mL solution in reaction buffer (20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA)). 6-10 equivalents of tris(2-carboxyethyl)phosphine (TCEP) per 10 mM of antibody or DTT were added to ANT4044-IgG1 in reaction buffer (6-10 mg / mL, 40°C). The antibody concentration was adjusted to 5 mg / mL by dilution with reaction buffer. The reduction mixture was incubated at 37-40°C for 1 hour. The reduction mixture was cooled to 22°C before adding the reagents. 5.6-8 equivalents of ThioBridge™-PEG(6u)-DOTA in acetonitrile were added to the mixture, which was further diluted to 4 mg / mL with reaction buffer. The percentage of acetonitrile in the mixture was 5%. The reaction mixture was incubated at 22°C for up to 22 hours. Buffer exchange and removal of excess reagents were performed by ultracentrifugation at 14,000rcf using Vivaspin 20 filters (30kDa MWCO, PES membrane, Generon). Samples were exchanged 7-9 times into Dulbecco's PBS pH 7.2-7.5 using Vivaspin 20 filters (30kDa MWCO, PES membrane, Generon). Antibody-ThioBridge™ DOTA conjugate concentrations were measured by UV-vis, corrected to 4.0mg / mL using Dulbecco's PBS pH 7.2-7.5, sterile filtered (0.22μm cellulose acetate filter) and stored at -80°C.

[0277] Lysine-DOTA conjugation evaluation: ANT4044-IgG1 was prepared as a 6 mg / mL solution in 0.1 M NaHCO3 and 20 mM ethylenediaminetetraacetic acid (EDTA), pH 8-9 (reaction buffer). 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester hexafluorophosphate trifluoroacetate (NHS-DOTA reagent) was then prepared at 5.0 mg / mL in Dulbecco's PBS pH 7.2-7.5. 10-25 equivalents of NHS-DOTA reagent solution were added and reaction buffer was added to correct the antibody concentration to 4.0 mg / mL. Incubated at 22 °C for 2-3 h. It was then quenched by the addition of 0.2 M sodium acetate, pH 5.5 (4:1 v / v) and ultracentrifuged at 14,000 rcf using a Vivaspin 20 filter (30 kDa MWCO, PES membrane, Generon). The dilution and ultracentrifugation were repeated two more times. The buffer was then exchanged four times into Dulbecco's PBS pH 7.2-7.5 using a Vivaspin 20 filter (30 kDa MWCO, PES membrane, Generon). The antibody-DOTA conjugate concentration was measured by UV-vis, corrected to 4.0 mg / mL using Dulbecco's PBS pH 7.2-7.5, sterile filtered (0.22 μm cellulose acetate filter), and stored at -80 °C. Small scale reactions and purifications were initially performed to identify suitable conjugation conditions. Analytical SEC and analytical LC-MS methods were developed to confirm the extent of conjugation, purity, and any residual reagents present. Conjugation was found to be effective for both reagents. Neither reagent resulted in aggregation during or after conjugation. The lysine conjugate showed a broader range of different DOTA-loaded species and required higher amounts for LC-MS analysis than the ThioBridge™ conjugate. All samples tested were shown by LC-MS to have an average DAR of 4.0-4.2 (ThioBridge™ conjugate) and 3.8-4.9 (lysine conjugate).

[0278] Example 3: Pharmacokinetic analysis of antibodies that bind to PSMA Using methodology similar to that described in Example 1, the serum half-life of the following antibodies was evaluated: J591 IgG lysine-DOTA conjugate (control antibody for binding to PSMA), ANT4044 lysine-DOTA conjugate (ANT4044-K-DOTA). , ANT4044-A2 lysine DOTA conjugate (ANT4044-A2-K-DOTA), ANT4044 with amino acid substitutions in the FcRn binding region, lysine DOTA conjugate (ANT4044-FcRn-K-DOTA), and ANT4044 with amino acid substitutions in the FcRn and Fc gamma receptor binding regions, lysine DOTA conjugate (ANT4044-FcRg-K-DOTA). The results are shown in Figure 1.

[0279] Figure 2 shows the mean area under the curve (AUC, top) and clearance (CL, bottom) for each antibody tested. Error bars represent the standard error of the mean.

[0280] Example 4: Biodistribution and Tumor Accumulation Studies Quantitative analysis of comparative targeting of different antibodies of the invention to LNCaP cells in a mouse model was assessed.

[0281] Radiolabeling of antibodies and TLC analysis. The test antibody is 1.ANT4044-IgG1+DOTA (“JN005”) 2.ANT4044-A2-IgG1+DOTA (“JN008”) 3.ANT4044-IgG1(FcRn)+DOTA(“JN007”) 4. ANT4044-IgG4 (FcRn / FcR gamma) + DOTA ("JN006") 5. HuJ591 IgG1+DOTA (control for binding to PSMA).

[0282] All antibodies were diluted in 0.1M pH 5.5 ammonium acetate buffer at 200-fold excess over the biomolecule. 64Cu was incubated at room temperature for 45 min. A sample of each solution was taken and mixed 1:1 with 50 mM EDTA. 5 μL of each solution was spotted onto TLC paper (Agilent iTLC-SG Glass microfiber chromatography paper impregnated with silica gel) and run with 50:50 H2O:ethanol. Plates were then imaged with a radioisotope phosphorescent screen on a Carestream MSFX imaging system. When necessary, unbound copper was removed using a 7 K MWCO Zeba Spin column (Thermo Scientific) according to the manufacturer's protocol. All samples showed >95% labeling.

[0283] Control experiments were performed to monitor the elution behavior of free Cu-64 and Cu-64 bound to EDTA for quality control.

[0284] Tumor Initiation and Growth Eight-week-old male Balb / C nude mice were injected subcutaneously (27G needle) with 5x10^6 LNCaP cells in 100μL phosphate-buffered saline in the right flank of each mouse.

[0285] Antibody solutions were injected via the tail vein (29G) and then the mice were imaged using a Siemens Inveon PET-CT device or blood was collected by tail snip and activity was measured by gamma counter for blood concentration analysis at the indicated time points.

[0286] Imaging protocol Mice were anesthetized with isoflurane (IsoFlo, Abbott Laboratories) at a dose of 2% in a closed anesthesia induction chamber. Mice were monitored using eye and paw reflexes to confirm deep anesthesia. After the mice were deeply anesthetized, they were placed on a suitable animal bed where an anesthetic air mixture (1%) was delivered to the nose and oral cavity through a nose cone. Physiological monitoring (respiration using a sensor probe) was performed using an animal monitoring system. (BioVet™ system, m2m Imaging, Australia) was used throughout the experiment. Images were acquired using a Siemens Inveon PET-CT scanner after intravenous tail vein injection of antibodies.

[0287] Syringes were filled with the radioisotope solution (approximately 150 μL) and the activity in the syringes was measured using a dose calibrator (Capintec CRC-25) with a calibration factor of 35. The activity remaining in the syringes after tail vein injection was measured using the same dose calibrator and the total volume injected into each mouse was calculated.

[0288] Calibration of the PET / CT scanner was performed using an in-house constructed phantom containing a dose of Cu-64 solution as the radiation source.

[0289] Mice were positioned on the scanner bed (n=4 per scan using an in-house developed bed) and micro-CT scans for anatomical co-registration were acquired. CT images of the mice were acquired via an X-ray source with the voltage set at 80 kV and the current set at 500 μA. Scans were performed at low magnification and binning factor 4 using a 360° rotation with 120 rotation steps. The exposure time was 230 ms and the effective pixel size was 106 μm. CT images were reconstructed using Feldkamp reconstruction software (Siemens). After CT imaging, PET scans were acquired 8 h, 24 h and 48 h after injection of the radiotracer using a 30-60 min static acquisition. PET images were reconstructed using an ordered-subset expectation maximisation (OSEM2D) algorithm and analysed using Inveon Research Workplace software (IRW 4.1) (Siemens), which allows for fusion of CT and PET images and definition of regions of interest (ROIs). The CT and PET data sets of each individual animal were aligned using IRW software (Siemens) to ensure good overlay of organs of interest. Three three-dimensional ROIs were placed within the whole body and all organs of interest, such as the heart, kidneys, lungs, bladder, liver, spleen, intestine and tumour, to delineate the organs using morphological CT information. Activity per voxel was converted to nci / cc using conversion factors obtained by scanning a cylindrical phantom filled with Cu-64 of known activity to account for the PET scanner efficiency. Activity concentrations were then calculated as the mean per cm of tissue. 3 The results were expressed as percent of the decay-corrected injected activity per gram, which can be approximated as the percentage of injected dose / g (%ID / g).

[0290] The tumor-to-blood ratio was then calculated as the activity detected in the tumor relative to the activity detected in the blood.

[0291] result Mice were imaged at 8, 24 and 48 hours post-injection, after which organs were harvested for gamma counting and quantification of organ distribution.

[0292] Regions of interest were drawn around the tumor margins (delineated by CT scan) and antibody concentrations were calculated for each mouse (based on % injected dose) in the imaging studies. Figures 3-6 and 8 show organ accumulation measured in vivo and ex vivo (by gamma counter). Variation in amounts between in vivo and ex vivo arises due to ROI and background signal for the in vivo plots. ns P>0.05, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

[0293] FIG. 7 shows blood levels of antibody up to 5 days after injection.

[0294] In vivo imaging demonstrated tumor accumulation and long-term localization (>2 days) for all antibodies There was no statistically significant difference between the different antibodies, and the tumor load was approximately 5% ID / g after 48 hours.

[0295] Ex vivo analysis showed a 48 hour biodistribution for all antibodies, at which point tumor deposits had the highest concentration of antibody, followed by the liver, spleen and lungs.

[0296] JN005 shows lower liver and spleen accumulation than other mutants (except J591), which is evident even at much longer circulation times (approximately 10% ID / g of JN005 still circulating at 120 hours).

[0297] Pharmacokinetic evaluation was performed by taking blood samples after 120 hours.The most notable observation was the rapid clearance of JN007 compared to the other antibodies.

[0298] Blood samples were also used to calculate the tumor:blood ratio of the antibodies. The tumor:blood ratio (in vivo:tail bleed) for each of the antibodies was determined for the 8, 24 and 48 hour time points. The results are shown in Figure 9. Figure 10 shows the tumor:blood ratio (ex vivo:ex vivo) at 48 and 120 hours.

[0299] Tumor:blood ratios were significantly higher at all time points for antibodies JN006 (ANT4044-IgG4(FcRn / FcR gamma)+DOTA) and JN007 (ANT4044-IgG1(FcRn)+DOTA) compared to antibodies JN005 (ANT4044-IgG1+DOTA) and J591. The ratios at 120 hours were particularly striking, with tumor:blood ratios for the FcRn binding engineered and FcRn / Rf gamma receptor binding engineered antibodies being approximately 200-fold higher than the ratios for the unengineered antibodies.

[0300] Consideration Although some of the tested antibodies had significantly decreased serum half-lives (and increased clearance), the amount of total antibody accumulating in the tumor was not statistically different. These results are surprising because they indicate that the tumor burden of each of the antibodies was the same, and that although FcRn and Fc gamma receptor modifications significantly increased the clearance and decreased the serum half-life of the JN007 and JN006 antibodies, all of the antibodies had similar binding affinity for their target epitopes, and all of the antibodies had similar abilities to be delivered to the target site.

[0301] The results show that modification of the FcRn or FcRn and Fc gamma receptor binding domains of radiolabeled antibodies has significant utility in reducing the amount of radioisotope in circulation without affecting the therapeutic potential of the antibody with respect to its ability to accumulate in tumors. This has many advantages, including reducing many of the toxic effects (including blood toxicity, bone uptake and bone marrow irradiation) that would otherwise result from longer residence of the radioisotope in the circulation.

[0302] Example 5: Preparation of Exemplary Antibodies of the Invention177 Lu Imaging and Radiotherapy Efficacy Study Test Article 1.TXP02-JN007(ANT4044-FcRn-K-DOTA) 2.TXP02-JN005(ANT4044-K-DOTA) 3. PSMA-617 (PSMA-binding peptide) Phase 1 Chemistry: Imaging Studies Sign summary: JN007, JN005.

[0303] ANT4044 is an anti-PSMA antibody described herein. -FcRN is a modified form of an antibody in which the FcRn binding region of the heavy constant chain has been modified to reduce serum half-life. ANT4044-FcRn-K-DOTA is ANT4044-FcRN conjugated to the chelator DOTA via a lysine residue.

[0304] 150 μg of ANT4044-FcRn was labeled with 500 MBq of Lu-177 by incubation at 37° C. for 2 hours. The labeling efficiency was 81%. The reaction mixture was purified on a NAP-5 column into PBS, after which the labeling efficiency was 98%. Fractions containing labeled antibody were collected. Doses containing 10 μg, 100 μg, and 370 μg, respectively, labeled with 20 MBq of Lu-177 were prepared by adding the appropriate amount of unlabeled ANT4044-FcRn antibody and PBS.

[0305] 500 μg of ANT4044 antibody was labeled with 100 MBq of Lu-177 by incubation for 2 hours at 37° C. The labeling efficiency was 98.2%. The preparation was diluted in PBS and used without further purification.

[0306] Phase 2 Chemistry: Efficacy Studies Label summary: PSMA-617, ANT4044-FcRn. 6 μg (4 nmol) of PSMA-617 (6 μl of 1 mg / ml solution in water) was labeled with 200 MBq Lu-177 in 0.1 M ammonium acetate buffer (pH 5.5) and heated to 95° C. for 10 min. The labeling efficiency was 97.6%. The preparation was diluted in PBS and used without further purification.

[0307] 300 μg ANT4044-FcRn was labeled with 120 MBq Lu-177. The labeling efficiency after 2 h incubation at 37° C. was 96.5%. The formulation was added in 5 μl It was quenched by adding 0.1 M EDTA, diluted in PBS and used without further purification.

[0308] Phase 1 - Imaging Study Design 1. For Phase 1 trials, select tumors of appropriate size (150-300 mm 3 ) were placed into 4 groups of n=3 with the following doses during the study: Group 1: 10 μg in 200 μl 177 Lu]TXP02-ANT4044-FcRn, intravenous (iv) injection into the lateral tail vein. b. Group 2: 100 μg in 200 μl 177 Lu]TXP02-ANT4044-FcRn, IV injection into the lateral tail vein. c. Group 3: 370 μg in 200 μl 177 Lu]TXP02-ANT4044-FcRn, IV injection into the lateral tail vein. d. Group 4: 100 μg in 200 μl 177 Lu]TXP02-ANT4044, IV injection into the lateral tail vein. 2. Antibody biodistribution was assessed 4, 24 and 48 hours after antibody injection via SPECT / CT imaging. a. Whole-body static SPECT images were acquired, followed by whole-body CT with respect to anatomical references. b. SPECT scan time was 40 minutes per scan, and CT scan time was 12 minutes. c. Animals were imaged three at a time in a multi-mouse hotel. 3. Tumor volumes were assessed three times a week by caliper measurements. Animals were examined for any adverse effects and weighed regularly. 4. For select animals, tissues were collected for further ex vivo analysis. 5. The remaining animals were killed and the carcasses were discarded.

[0309] Phase 2 - Efficacy Study Design 1. For efficacy studies, select tumors of appropriate size (150-300 mm 3 ) were placed into 3 groups of n=6 with the following doses during the study: Group 1: 50 μg in 200 μl 177 Lu]TXP02-ANT4044-FcRn, IV b. Group 2: 200 μl of 600 ng [ 177 Lu]PSMA-617, IV c. Group 3: 200 μl PBS, IV 2. After test drug injection, tumor size was assessed by caliper measurement three times a week for three weeks. 3. Animals were examined for any adverse effects and weighed regularly. 4. Blood samples were taken from animals in groups 1 and 2 by tail puncture method at 0.5, 4, 8, 24, 48, 72, 96, 120 hours and assessed by gamma counting. 5. Whole blood samples were weighed and then counted in a gamma counter with reference standards. 6. In the initial study, the objective was to monitor tumor growth for 5 weeks after treatment. This time frame was 177 Lu]TXP02-ANT4044-FcRn mice because of radiation injury observed in some of the mice. 7. For select animals, tissues were collected for further ex vivo analysis. 8. The remaining animals were killed and the carcasses were discarded.

[0310] Results and Discussion FIG. 11 shows the results of the [ 1771 is an exemplary image of the distribution of [Lu]TXP02-ANT4044-FcRn.

[0311] Figure 12 shows the [ 177 Lu]TXP02-ANT4044-FcRn radioactivity levels.

[0312] Figure 13 is a plot of the tumor growth determined in this study. ANT4044-FcRn-DOTA-Lu treatment significantly inhibited tumor growth as evidenced by no change in tumor volume at day 14 compared to day 0. In the control (PBS) group, there was an overall increase in tumor volume, with tumors becoming significantly larger at days 9, 12, and 14 when compared to the corresponding times in the ANT4044-FcRn-DOTA-Lu treated group.

[0313] Labelling of both the test (JN007) and comparator (JN005) antibodies was successful (>96% labelling efficiency) and a maximum specific activity of 2MBq / μg was achieved.

[0314] Imaging studies demonstrated uptake of both test and comparative antibodies into LNCaP tumor xenografts (see Table 2).

[0315] As estimated by a small ROI drawn within the left ventricle of the heart, blood clearance was faster for the JN007 antibody for all groups at 48 hours post-injection compared to the same time point for the JN005 antibody (see Table 3).

[0316] [Table 7]

[0317] [Table 8]

[0318] FIG. 14 is a plot of the tumor:blood ratio in mice following administration of the JN007 antibody (an anti-PSMA antibody of the invention engineered to reduce FcRn binding, also called K-DOTA-Lu antibody-HuX592R-DOTA-Lu177).

[0319] Compared to control mice (JN005, i.e., which received the anti-PSMA, K-DOTA-Lu antibody HuJ591-DOTA-Lu177 without modifications in the FcRn binding region), mice that received the FcRn modified antibodies had a higher ratio of antibodies in the tumor compared to the blood.

[0320] Example 6 Test antibody 1. HuX592R (ANT4044-FcRN). 2.HuJ591(ANT4044).

[0321] Radiolabeling of antibodies and TLC analysis All antibodies were diluted in 0.1M pH 5.5 ammonium acetate buffer at 50-fold or 100-fold excess of each biomolecule for HuX592R and HuJ591. 177 The solutions were incubated with Lu for 45 min at room temperature. A sample of each solution was taken and mixed 1:1 with 50 mM DTPA. 5 μL of each DTPA-incubated sample or neat solution was spotted onto TLC paper (Agilent iTLC-SG Glass microfiber chromatography paper impregnated with silica gel) and flushed with 50:50 HO:ethanol. Detection of radiolabeled species transfer was achieved using an Eckert and Ziegler Mini-Scan and Flow-Count system. All samples showed >90% labeling. Control experiments were performed to determine the free 177 Lu and DTPA bound 177 The elution behavior of Lu was monitored for quality control.

[0322] cell binding Lu-labeled constructs HuX592R and HuJ591 were assessed for cell binding.

[0323] In summary, 1.25x10 in 0.100mL PBS 5 PC-3 tumor cells (negative control) or 1.25x10 5 Eppendorf tubes containing LNCaP tumor cells are incubated with 5 μL (0.030 MBq) of labeled antibody at 37° C. Incubation was stopped at the following time points for analysis: 1 h, 2 h, 4 h and 24 h.

[0324] Determination of unbound fraction. At the end of each incubation period, the Eppendorf tubes are centrifuged at 500g for 5 minutes. The supernatants containing free Lu-177 antibody are collected in separate tubes and counted using gamma analysis. The pellets containing cells bound to the Lu-177 labeled antibody are washed with 0.200 mL of PBS solution and centrifuged three times at 500g for 5 minutes. The supernatants from each wash are collected and counted, and the values ​​are combined with the collected incubation supernatants to obtain the total free unbound antibody.

[0325] Determination of surface-bound fraction. The pellet was resuspended in 0.1 mL PBS pH 4.0 for 20 min at ice-cold temperature. The Eppendorf tubes were then centrifuged at 500 g for 5 min at 4° C. and the supernatant was collected for counting. After three further washes with ice-cold PBS pH 4.0, the supernatant was collected for gamma counting.

[0326] Determination of internalization fraction. After washing and centrifugation, the cell pellet was counted as the internalized fraction.

[0327] Protein concentration Cells were solubilized with 1 M NaOH and protein concentrations were determined.

[0328] calculation Lu-177 antibody binding to tumor cells (surface-bound and internalized fraction) and unbound antibody were calculated and reported as a percentage of total radioactivity incubated per mg of protein.

[0329] animal Six-week-old healthy male Balb / C nude mice (approximately 20 g) were cultured at 100°C for 12 h at 4 °C in a Western Mice were obtained from the University of Maryland (Australia) and used in this study. Mice were monitored for one week prior to the study to allow for acclimation to the environment prior to cell injection. All animals were provided with free access to food and water before and during the experiment.

[0330] Administration Syringes were filled with radiolabeled antibody solution (approximately 100 μL) and activity in the syringes was measured using a dose calibrator (Capintec CRC-25) with a calibration factor of 35. Activity remaining in the syringes after tail vein injection was measured using the same dose calibrator and the total volume injected into each mouse was calculated.

[0331] Tolerability Healthy male Balb / C nude mice (n=3 per treatment cohort) were 177 Lu-labeled HuX592R was injected (29G, tail vein injection in approx. 100 μL saline) a total of three times, 7 days apart, to give injection doses of 6, 9 or 12 MBq at a bolus of approx. 100 μg per mouse. Mice's health was then monitored after injection, and all mice were killed 28 days after the first injection, with organs fixed in PFA, transferred to 30% sucrose, and disintegrated, if necessary, before being cryopreserved for future analysis.

[0332] Distribution in the body Healthy male Balb / C nude mice (n=3 per cohort) were 177 Lu-labeled constructs were injected (29G, tail vein injection saline) to give an injection dose of 6MBq at a bolus of 100 (HuX592R) or 140 (HuJ591) μg per mouse. Animals were then sacrificed at 24, 48 and 72 hours (for HuX592R) and 72 hours (for HuJ591) post-injection. Blood was sampled and tissues were collected, washed of excess blood and weighed for ex vivo analysis. Radioactivity in tissues was measured using a Perkin PerkinElmer 2480 Automatic gamma counter. The gamma counter was177 Calibration was performed using known samples of Lu, and the measured activity was expressed as %ID / g of tissue weight based on injected activity.

[0333] Tumor Initiation and Growth For autoradiography and treatment studies, 8-12 week old male Balb / C nude mice were inoculated with 4x10 6 LNCaP cells were injected subcutaneously (27G needle) into the right flank of each mouse. There was no evidence of ulceration at the time of cell injection; animals were closely monitored and tumors were measured with calipers and remained well apart from solid tumor growth. Tumor growth was sporadic, as is often observed with LNCaP tumors, with tumors growing between 100-200 mm 3 Once the .times.1 ...

[0334] Autoradiography Tumor-bearing male Balb / C nude mice (n=2 per cohort) were 177 Lu-labeled constructs were injected (29G, tail vein injection in approximately 100 μL saline) to give an injection dose of 6 MBq at a bolus of 100 (HuX592R) or 140 (HuJ591) μg per mouse. Animals were then sacrificed 7 days after injection and tumor samples were snap frozen in an isopentane-dry ice slurry before being embedded in OCT compound for sectioning. 20 μm sections were collected on slides and air-dried before being exposed for approximately 3.5 hours on a phosphor screen in a closed cassette. The sections were then transferred to the Amersham Typhoon Using Phosphorimage, sensitivity setting 1000 177 Images were acquired using a pixel size of 50 gm (scan time 20-30 min) optimized for Lu. Images were analyzed using ImageQuant TL software.

[0335] Treatment trials Tumor-bearing male Balb / C nude mice (n=6 per cohort) were 177Lu-labeled constructs were injected (29G, tail vein injection in approx. 100 μL saline) to give injection doses of 6 or 8 MBq HuX592R or 6 MBq HuJ591 at boluses of 100 μg HuX592R or 140 μg HuJ591 per mouse. Tumor growth in response to treatment was monitored over a 90-day period compared to vehicle-only controls.

[0336] result Both HuX592R and HuJ591 177 The success of the Lu labeling was demonstrated. 177 Lu-labeled constructs were directly subjected to cell binding, tolerability, biodistribution and therapeutic efficacy studies. Used in contact.

[0337] cell binding 177 Binding of Lu-labeled HuX592R and HuJ591 was assessed to LNCaP (PSMA+) and PC3 (PSMA-) cell lines. Unbound, surface-bound, and cell pellet-bound fractions were assessed 1, 2, and 4 hours after adding constructs to the cells. Cell assays showed that both HuJ591 and HuX592R showed enhanced accumulation in PSMA-expressing cell lines, with the highest concentrations observed in the cell pellets indicating successful internalization during the assay period (all time points). Minimal binding / internalization was generally observed in the PSMA-negative cell line (PC3), indicating that binding is receptor-dependent and that the label does not significantly interfere with antibody binding to the receptor.

[0338] Tolerability In healthy male Balb / c nude mice (n=3 per cohort) 177 The tolerability of Lu-labeled HuX592R was evaluated at three dose levels, 12, 9 and 6 MBq, administered three times at weekly intervals. Animal health was evaluated for 4 weeks, including the 3rd week of therapeutic administration, by animal weight and animal score sheet. In this study, the dose of 12 MBq was found to be too high, so all studies were performed using doses of 6 MBq and 8 MBq.

[0339] Distribution in the body In healthy male Balb / c nude mice (n=3 per cohort) 177 Biodistribution of Lu-labeled constructs was assessed at 24, 48, and 72 hours post-dose for HuX592R (Figure 15A), and only at 72 hours post-dose for HuJ591 (Figure 15B). Results showed a significantly higher circulation time for HuJ591 compared to HuX592R, with comparable lower levels of accumulation in clearance organs such as the liver and spleen, suggesting a more rapid clearance by the system.

[0340] Autoradiography 177 Lu-labeled constructs were administered to tumor-bearing male Balb / C nude mice. Animals were sacrificed 1 week after injection, tumors were frozen in OCT, sectioned, and autoradiography images were acquired. Overall results were consistent with tumor accumulation and penetration by both constructs, with higher accumulation of HuJ591 compared to HuX592R at this time point. Furthermore, distribution of the radiotherapeutic appeared to be well dispersed throughout the tumor for both antibodies.

[0341] Treatment trials Male Balb / C nude mice bearing flank LNCaP xenograft tumors were assigned to treatment cohorts and received three doses, one week apart, of one of the following: 1. Vehicle control 2.6MBq[ 177 Lu]-HuX592R 3.8MBq[ 177 Lu]-HuX592R 4.6MBq[ 177 Lu]-HuJ591

[0342] Animal health, as assessed by tumor regression (Figure 17) and animal weight, was monitored for 100 days. Results showed significant tumor growth inhibition for both HuX592R (Figure 16) in 2 mice in the HuX592R 8MBq cohort, and 4 mice in the HuJ591 cohort (not shown) showed complete tumor regression, where tumor burden could not be measured by caliper. Thirty-seven days after treatment initiation (n=6 for all cohorts, last data point allowing statistical comparison), all three treatments, 6MBq HuX592R (p 0.0105), 8MBq HuX592R (p 0.0086) and 6MBq HuJ591 (p 0.0056), showed a significant tumor growth inhibition of 2.5% of the HuX592R cohort (p 0.0056). There was a significant reduction in tumor growth compared to the control cohort as determined by one-way analysis of variance.

[0343] None of the treatment groups showed any significant health problems as assessed by changes in body weight.

[0344] Mouse survival (Figure 17) was statistically longer (p<0.05 by Mantel-Cox test) in all three treatment cohorts (median survival>study period) compared to the control cohort (median survival 83.5 days). Upward check marks indicate animals that were euthanized due to non-tumor / health-related issues.

[0345] Both formulations (HuJ591 at 6MBq) and HuX592R (both 6MBq and 8MBq) showed efficacy against PSMA-expressing tumors. Both HuJ591 (at 6MBq) and HuX592R (at 8MBq) had no treatment / tumor-related deaths during the 100 days of the study, and 2 mice in the HuX592R 8MBq and 4 mice in the HuJ591 group showed complete regression of tumors (not measurable by caliper). In terms of survival plots, all treatment groups with antibodies were statistically better than the control group.

[0346] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. An antibody having reduced FcRn binding affinity compared to a wild-type antibody of class IgG, a heavy chain constant region, in which one or more amino acid residues at positions His310, His433, His435 and Ile253, numbered according to the EU numbering system, differ from those present in a wild-type antibody of said class IgG; Including, The antibody specifically binds to prostate specific membrane antigen (PSMA) and (i) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3 or 19; and (ii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 33, a CDR2 comprising the sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the sequence set forth in SEQ ID NO: 35; An antibody comprising an antigen-binding domain comprising:

2. The antibody described in claim 1, wherein the amino acid residues at His310 and His435, as numbered by the EU numbering system, differ from the residues present in a wild-type antibody of class IgG.

3. The antigen-binding domain, (i) a VH comprising a framework region (FR) 1 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO:25, a FR2 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO:26, a FR3 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO:27, and a FR4 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO:28; (ii) a VL comprising an FR1 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 41, an FR2 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 42, and an FR3 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 43, and an FR4 comprising a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 44; (iii) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 25, FR2 comprising the sequence set forth in SEQ ID NO: 26, FR3 comprising the sequence set forth in SEQ ID NO: 27, and FR4 comprising the sequence set forth in SEQ ID NO: 28; (iv) a VL comprising an FR1 comprising the sequence set forth in SEQ ID NO: 41, an FR2 comprising the sequence set forth in SEQ ID NO: 42, an FR3 comprising the sequence set forth in SEQ ID NO: 43, and an FR4 comprising the sequence set forth in SEQ ID NO: 44; or (v) VH comprising FR1 comprising the sequence shown in SEQ ID NO: 25, FR2 comprising the sequence shown in SEQ ID NO: 26, FR3 comprising the sequence shown in SEQ ID NO: 27, and FR4 comprising the sequence shown in SEQ ID NO: 28; and VL comprising FR1 comprising the sequence shown in SEQ ID NO: 41, FR2 comprising the sequence shown in SEQ ID NO: 42, and FR3 comprising the sequence shown in SEQ ID NO: 43, and FR4 comprising the sequence shown in SEQ ID NO:

44. The antibody of claim 1, further comprising at least one of the following:

4. The antibody described in claim 1, comprising an antigen-binding domain comprising a VH having an sequence as set forth in SEQ ID NO: 4 or 20 and a VL having an sequence as set forth in SEQ ID NO:

36.

5. The antibody described in claim 1, wherein the antibody contains one or more amino acid substitutions that reduce the affinity of the antibody for one or more Fc gamma receptors compared to a wild-type antibody of class IgG.

6. The antibody of claim 1, wherein the antibody comprises one or more amino acid substitutions that increase the stability of the CH1-CH2 hinge region in the antibody compared to a wild-type antibody of class IgG.

7. The antibody of claim 1, wherein the heavy chain constant region of the antibody comprises amino acid substitutions at positions Ser228 and Leu235 of the heavy chain constant region, as numbered according to the EU numbering system.

8. The antibody of claim 7, wherein the amino acid substitutions at positions Ser228 and Leu235, as numbered by the EU numbering system, are Ser228Pro and Leu235Glu.

9. The antibody described in claim 1, wherein the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO:50 or SEQ ID NO:

51.

10. The antibody comprising: (i) SEQ ID NO: 53 and SEQ ID NO: 57; (ii) SEQ ID NO: 54 and SEQ ID NO: 57; (iii) SEQ ID NO: 55 and SEQ ID NO: 57, or (iv) SEQ ID NO: 56 and SEQ ID NO: 57 The antibody of claim 1, comprising the amino acid sequence shown in 11. The antibody of claim 1, further comprising a non-proteinaceous agent conjugated thereto, the non-proteinaceous agent comprising a therapeutic or diagnostic agent.

12. The antibody of claim 11, wherein the therapeutic agent is a cytotoxin.

13. The antibody described in claim 11, wherein the therapeutic agent or diagnostic agent is a radioactive element.

14. The antibody of claim 11, wherein the non-protein agent is a radioactive isotope.

15. The radioisotope is actinium-225 (225Ac), astatine-211 (211At), bismuth-212 and bismuth-213 (212Bi, 213Bi), copper-64 and copper-67 (64Cu, 67Cu), gallium-67 and gallium-68 (67Ga and 68Ga), indium-111 (111In), iodine-123, -124, -125 or -131 (123I, 124I, 125I, 131I), lead-212 (212Pb), lutetium-177 (177Lu), radium-223 (223Ra), samarium-153 ( 15. The antibody of claim 14, wherein the ion is selected from the group consisting of Zn-Al-Fe-SiO2 ( 153 Sm), Scandium-44 and Scandium-47 ( 44 Sc, 47 Sc), Strontium-90 ( 90 Sr), Technetium-99 ( 99m Tc), Yttrium-86 and Yttrium-90 ( 86 Y, 90 Y) and Zirconium-89 ( 89 Zr).

16. The antibody of claim 15, wherein the radioisotope is selected from actinium-225 ( 225 Ac), astatine-211 ( 211 At), lutetium-177 ( 177 Lu) and zirconium-89 ( 89 Zr).

17. A pharmaceutical composition comprising an antibody described in any one of claims 1 to 16 and a pharma- ceutically acceptable excipient.

18. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 16 for use in treating prostate cancer in a subject in need thereof.

19. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 16 for use in the diagnosis, monitoring or prognosis of a disease, disorder or infection in a subject.

20. The pharmaceutical composition for use according to claim 19, wherein the antibody is conjugated to a radiolabel.

21. The radiolabel is actinium-225 (225Ac), astatine-211 (211At), bismuth-212 and bismuth-213 (212Bi, 213Bi), copper-64 and copper-67 (64Cu, 67Cu), gallium-67 and gallium-68 (67Ga and 68Ga), indium-111 (111In), iodine-123, -124, -125 or -131 (123I, 124I, 125I, 131I), lead-212 (212Pb), lutetium-177 (177Lu), radium-223 (223Ra), samarium-153 (153 21. The pharmaceutical composition for use according to claim 20, wherein the metal ions are selected from the group consisting of Scandium-44 and Scandium-47 ( 44 Sc, 47 Sc), Strontium-90 ( 90 Sr), Technetium-99 ( 99m Tc), Yttrium-86 and Yttrium-90 ( 86 Y, 90 Y) and Zirconium-89 ( 89 Zr).

22. The pharmaceutical composition for use according to claim 21, wherein the radiolabel is selected from gallium-67 and gallium-68 ( 67 Ga and 68 Ga), iodine-124 ( 124 I) and zirconium-89 ( 89 Zr).

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