Methods for treating cancers that express PDGFR alpha

Abstract

The present invention relates to a composition comprising an olaratumab antibody conjugated to a therapeutic radioisotope for the treatment of cancer expressing platelet-derived growth factor receptor alpha (PDGFR-alpha), and to a method of using the same.

Description

Technical Field

[0001] The present invention relates to proteins and compositions for the treatment of cancers that express platelet-derived growth factor receptor alpha (PDGFR alpha), and methods of using the same.

Background Art

[0002] Soft tissue sarcoma (STS) originates from soft tissues or connective tissues such as fat, muscle, nerve, fibrous tissue, blood vessels, or deep skin tissue. STS is a heterogeneous disease with relatively few effective regimens.

[0003] Therefore, there is a need for new and improved methods and reagents for the treatment of STS and related cancers.

[0004] Any reference to prior art herein does not admit, or suggest, that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art is regarded as relevant, and / or reasonably foreseeable to be combined with other prior art, by a person skilled in the art.

Summary of the Invention

[0005] The present invention relates to reagents and methods for treating cancers that express platelet-derived growth factor receptor alpha (PDGFR alpha).

[0006] In a first aspect, the present invention provides an olaratumab antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is - conjugated to a radioisotope for treating, preventing, or minimizing the progression of cancer characterized by, or associated with, the expression of PDGFR alpha.

[0007] It will be understood that an olaratumab antibody or an antigen-binding fragment thereof conjugated to a radioisotope may also be referred to as a radio-labeled olaratumab antibody or an antigen-binding fragment thereof.

[0008] In a second aspect, the present invention provides an olaratumab antibody or an olaratumab antibody bioconjugate suitable for radiolabeling with a therapeutic radioisotope.

[0009] Preferably, the olaratumab bioconjugate comprises an olaratumab antibody conjugated to any chelating or linker group suitable for further conjugation to a radioisotope. Optionally, the olaratumab bioconjugate is selected from olaratumab-TMT (6,6’’-bis[N,N’’,N’’’-tetrakis(carboxymethyl)aminomethyl)-4’-(3-amino-4-methoxyphenyl)-2,2’:6’,2’’-terpyridine), olaratumab-DOTA (1,4,7,10-tetraazacyclododecane-NN’,N’’(N’’’-tetraacetic acid), olaratumab-TCMC, olaratumab-DO3A, olaratumab-CB-DO2A, olaratumab-NOTA, olaratumab-diamsar, olaratumab-DTPA, olaratumab-CHX-A’’-DTPA, olaratumab-TETE, olaratumab-Te2A, olaratumab-HBED, olaratumab-DFO, olaratumab-DFOsq, olaratumab-DFO-NCS, and olaratumab-HOPO, or an olaratumab chelated to a chelating agent disclosed in WO2022 / 133537 or any other chelating agent described herein or known to those skilled in the art.

[0010] In any aspect, the olaratumab antibody or an antigen-binding fragment thereof preferably comprises an antigen-binding domain that competitively inhibits the binding of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO: 12.

[0011] In any aspect or embodiment herein, the olaratumab antibody or an antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 of an antigen-binding domain having a variable heavy chain defined by SEQ ID NO: 4, and LCDR1, LCDR2, and LCDR3 of an antigen-binding domain having a VL defined by SEQ ID NO: 12.

[0012] As used herein, the complementarity-determining region (CDR) of the antigen-binding protein of the present invention may be defined according to the IMGT, Chothia, or Kabat numbering system, or any other CDR numbering system known to those skilled in the art.

[0013] In any aspect or embodiment of the present invention, the olaratumab antibody or its antigen-binding fragment is an antigen-binding domain, (i) Complementarity Determination Region (CDR) 1, which contains at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence described in Sequence ID No. 1, and the sequence set in Sequence ID No. 2, which contains at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% of the sequence. VH, including CDR2 containing a sequence identical to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and CDR3 containing a sequence identical to at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, of the sequence described in Sequence ID No. 3. (ii) A VH containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequence to the sequence described in Sequence ID No. 4. (iii) CDR1 containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence is identical to the sequence described in Sequence ID No. 9, and CDR1 containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, and less than 80% of the sequence described in Sequence ID No. 10. VL, including CDR2 containing a sequence identical to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and CDR3 containing a sequence identical to at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, (iv) A VL containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequence to the sequence described in Sequence ID No. 12. (v) VH, containing CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3. (vi) VH containing the sequence described in sequence number 4, (vii) VL containing CDR1 containing the sequence described in SEQ ID NO: 9, CDR2 containing the sequence described in SEQ ID NO: 10, and CDR3 containing the sequence described in SEQ ID NO: 11. (viii) VL containing the sequence described in sequence number 12, (ix) VH containing CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3, and VL containing CDR1 containing the sequence described in SEQ ID NO: 9, CDR2 containing the sequence described in SEQ ID NO: 10, and CDR3 containing the sequence described in SEQ ID NO: 11, or (x) comprising an antigen-binding domain including VH containing the sequence described in SEQ ID NO: 4, and VL containing the sequence described in SEQ ID NO: 12.

[0014] Olaratumab antibody or its antigen-binding fragment is (i) Framework region (FR) 1 containing or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in SEQ ID NO: 5, at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in SEQ ID NO: 6 R2, FR3 containing or consisting of a sequence identical to or consisting of the sequence described in Sequence ID No. 7 by at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, respectively; VH, and FR4 containing or consisting of a sequence identical to or consisting of the sequence described in Sequence ID No. 8 by at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, respectively; (ii) Framework region (FR) 1 containing or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in Sequence ID No. 13; FR2 containing or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in Sequence ID No. 14. FR3, Sequence ID 16, contains or consists of a sequence that is at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence described in Sequence ID 15. VL may further include VL containing an FR4 that is at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence described in VL.

[0015] In further embodiments, the olaratumab antibody or its antigen-binding fragment is (i) VH, which includes or comprises a framework region (FR) 1 containing or comprising the sequence described in SEQ ID NO: 5, FR2 containing or comprising the sequence described in SEQ ID NO: 6, FR3 containing or comprising the sequence described in SEQ ID NO: 7, and FR4 containing or comprising the sequence described in SEQ ID NO: 8. (ii) A VL comprising a framework region (FR) 1 comprising or consisting of the sequence described in sequence number 13, FR2 comprising or consisting of the sequence described in sequence number 14, FR3 comprising or consisting of the sequence described in sequence number 15, and FR4 comprising or consisting of the sequence described in sequence number 16.

[0016] In any embodiment, the olaratumab antibody or its antigen-binding fragment contains a VH that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence described in SEQ ID NO: 4, and / or at least identical to the sequence described in SEQ ID NO: 12. VL contains approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences, and there is no sequence diversity between VH and VL and the sequences of SEQ ID NOs. 4 and 12 in CDR, and the antigen-binding protein retains its ability to bind to PDGFR alpha.

[0017] In any embodiment, the olaratumab antibody or its antigen-binding fragment comprises VH and / or VL, which each comprises substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues compared to the amino acid sequence described in SEQ ID NO: 4 or 12, wherein the amino acid substitutions, deletions, or additions are not present in the CDR, and the antigen-binding protein retains its ability to bind to PDGFR alpha.

[0018] As used herein, the terms HCDR1, HCDR2, and HCDR3 are understood to refer to variable heavy chain CDRs, the terms LCDR1, LCDR2, and LCDR3 are understood to refer to variable light chain CDRs, and the terms HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are understood to refer to heavy chain and light chain framework regions, respectively.

[0019] In the context of this specification, olaratumab antibody or its antigen-binding fragment is, (i) Single-domain antibody (sdAb), (ii) single chain Fv fragment (scFv), (iii) Dimer scFv (di-scFv), or (iv) It may be in the form of one of (ii) or (iii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of the antibody.

[0020] Furthermore, as described herein, olaratumab or its antigen-binding fragment is (i) Diabody, (ii) Triabody, (iii) Tetrabody, (iv)Fab, (v)F(ab')2, (vi)Fv, (vii) Bispecific antibodies or other forms of multispecific antibodies, (viii) It may be in the form of one of (i) to (vii) linked to the constant region of the antibody, Fc, or the heavy chain constant domain (CH)2 and / or CH3.

[0021] The variable weight and variable light regions of the antigen-binding domain are selectively linked via a linker.

[0022] In any embodiment, the antigen-binding domain is This may include FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a.

[0023] As defined herein, a linker may be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0024] In any aspect or embodiment of the present invention, olaratumab or its antigen-binding fragment may include a human constant region (for example, an IgG constant region such as the IgG1, IgG2, IgG3, or IgG4 constant regions or a mixture thereof). H and V L In the case of antibodies or proteins containing V H It can be linked to the heavy chain steady region, V L It can be linked to the light chain steady region.

[0025] In one example, olaratumab or its antigen-binding fragment contains the constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In another example, olaratumab or its antigen-binding fragment contains the IgG4 constant region with proline at position 241 (according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991)).

[0026] For example, olaratumab or its antigen-binding fragment contains a heavy chain constant region which includes a stabilized heavy chain constant region that contains a mixture of sequences, with or without a C-terminal lysine residue.

[0027] In further embodiments of any aspect of this specification, olaratumab or its antigen-binding fragment comprises an Fc region, the Fc region being engineered to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhancement of the ability to induce ADCC is conferred by mutation, deletion, or modification of amino acids in the Fc region that interacts with the Fc receptor.

[0028] In another embodiment, olaratumab or its antigen-binding fragment is - Having an increased in vitro or in vivo half-life, - Having an increased ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity, and / or - Includes an Fc region that has been manipulated to have reduced effector functionality.

[0029] Preferably, the olaratumab antibody is in the form of an antibody (i.e., a variable light chain and a variable heavy chain linked to the constant region of the antibody containing the heavy chain CH2 and / or CH3).

[0030] In further embodiments, olaratumab may include a heavy chain constant region as defined in SEQ ID NO: 17 and / or a light chain constant region as defined in SEQ ID NO: 18.

[0031] In certain embodiments, olaratumab may include the heavy chain described in SEQ ID NO: 19 and / or the light chain described in SEQ ID NO: 20.

[0032] In further embodiments, olaratumab may be in the form of an antibody and may contain one or more amino acid substitutions in the constant region to reduce the in vivo half-life of the antibody. Accordingly, the present invention provides an olaratumab antibody having substitutions in the CH2 and / or CH3 domains of the constant region and containing substitutions in one or more of the residues His310, His435, His436, and Ile253 (Kabat numbering), thereby altering the FcRn binding affinity and / or serum half-life of the antibody (for example, compared to olaratumab containing the natural CH2 and CH3 domains).

[0033] For example, the amino acid at positions 310 and / or 435 of the antibody may be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0034] Preferably, the residue at position 310 is selected from alanine, glutamic acid, or glutamine, or the amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine, or alanine. In another preferred embodiment, the antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0035] In further embodiments, the antibody also includes an amino acid substitution at residue Lys322. Preferably, the substitution is K322A.

[0036] In a particularly preferred embodiment, the olaratumab antibody comprises the substitutions K322A, H310A, and H435Q.

[0037] In preferred embodiments, the binding affinity of the modified antibody for FcRn and / or its serum half-life is reduced by at least about 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 preferred embodiments of the present invention, the binding affinity of the modified antibody for FcRn and / or its serum half-life is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0038] In any aspect or embodiment, the antibody may also contain amino acid substitutions (e.g., Ser228Pro and / or Leu235Glu) at residues corresponding to Ser228 and Leu235 in the constant heavy chain region.

[0039] According to the first aspect and any of its embodiments, the radiolabeled orlaratumab is conjugated to any radioisotope suitable for the treatment of tumors or cancer masses. Examples of suitable radioisotopes 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), 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 and radium-224 ( 223 Ra, 224 Ra), rhenium-186 and rhenium-188 ( 186 Re and 188 Re), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 (​90 Sr), terbium-149 and terbium-161 ( 149 Tb and 161 Tb), and Yttrium-90 ( 90 Y) is one example. In a preferred embodiment, the radionuclide conjugated with the antibody or a fragment thereof is actinium-225 or lutetium-177.

[0040] In any embodiment, the radiolabeled olaratumab antibody or its antigen-binding fragment is 89 Zr-, 225 Ac-, or 177 Lu-olaratumab is a radioactive isotope that is directly or indirectly conjugated to the olaratumab antibody.

[0041] In any embodiment or configuration, the radioisotope may be directly conjugated to olaratumab, for example, by halogenation of an amino acid residue. Preferably, the radioisotope of the radiolabeled olaratumab is indirectly conjugated to olaratumab or its antigen-binding fragment, for example, via a chelating agent or other binding moiety. For example, olaratumab is conjugated to a chelate portion selected from the group consisting of chelating agents described herein or known to those skilled in the art, such as 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-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, diamsal, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, and HOPO, WO2022 / 133537 (incorporated herein by reference), or other chelating agents described herein or known to those skilled in the art.

[0042] In another example, radiolabeled olaratumab or its antigen-binding fragment or olaratumab antibody bioconjugate may be conjugated to a bifunctional linker (e.g., bromoacetyl, thiol, succinimide ester, TFP ester, maleimide) or conjugated using any amine or thiol modification chemistry known in the art.

[0043] Preferably, the radiolabeled olaratumab antibody or its antigen-binding fragment is 177 Lu-Olaratumab or 177 Lu-labeled oraratumab 177 Lu is conjugated to olaratumab via a linker (for example, 177 Lu-DOTA-Olaratumab).

[0044] In a further embodiment, a method is provided for obtaining radiolabeled olaratumab according to a first embodiment of the present invention, wherein the method comprises radiolabeling an olaratumab bioconjugate or antibody according to a second embodiment.

[0045] In a further embodiment, a pharmaceutical composition is provided comprising, optionally in combination with a pharmaceutically acceptable excipient, the olaratumab antibody or its antigen-binding fragment, an olaratumab bioconjugate, or radiolabeled olaratumab as described herein.

[0046] Furthermore, nucleic acids or nucleic acid constructs encoding olaratumab antibodies or antigen-binding fragments thereof, or olaratumab bioconjugates, as described herein are provided.

[0047] Furthermore, a host or host cell containing the nucleic acid or nucleic acid construct of the present invention is provided.

[0048] In a fourth aspect, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, the method being: - This includes administering to subjects requiring it a radiolabeled olaratumab antibody or its antigen-binding fragment as described herein, This will treat, prevent, or minimize the progression of cancer in the target population.

[0049] In a further embodiment, the present invention provides a method for minimizing, reducing, or preventing tumor growth in a subject, the method being: - This includes administering to subjects requiring it a radiolabeled olaratumab antibody or its antigen-binding fragment as described herein, This minimizes, reduces, or prevents tumor growth in the target area.

[0050] In a further aspect, the present invention provides a method for minimizing, reducing, or preventing cancer metastasis in a subject, the method being: - This includes administering to subjects requiring it a radiolabeled olaratumab antibody or its antigen-binding fragment as described herein, This minimizes, reduces, or prevents cancer metastasis in the target area.

[0051] In a further embodiment, the present invention provides a method for increasing the survival time of a subject with cancer, the method being: - This includes administering to subjects requiring it a radiolabeled olaratumab antibody or its antigen-binding fragment as described herein, This increases the survival time of the target.

[0052] In another aspect, the present invention further provides the use of radiolabeled olaratumab or its antigen-binding fragments described herein in the manufacture of pharmaceuticals for the following: - To treat, prevent, or minimize the progression of cancer in the target population. - To minimize, reduce, or prevent tumor growth in the target area. - Minimizing, reducing, or preventing metastasis in the target, - To increase the survival time of individuals suffering from cancer.

[0053] In another aspect, the present invention further provides the use of radiolabeled olaratumab or its antigen-binding fragments described herein for the following purposes: - To treat, prevent, or minimize the progression of cancer in the target population. - To minimize, reduce, or prevent tumor growth in the target area. - Minimizing, reducing, or preventing metastasis in the target, - To increase the survival period of the subject.

[0054] In another aspect, the present invention further provides radiolabeled olaratumab or its antigen-binding fragments as described herein for use as follows: - To treat, prevent, or minimize the progression of cancer in the target population. - To minimize, reduce, or prevent tumor growth in the target area. - Minimizing, reducing, or preventing metastasis in the target, - To increase the survival period of the subject.

[0055] In any aspect or embodiment of the present invention, it will be understood that the amount of radiolabeled olaratumab or its antigen-binding fragment administered to a subject is a therapeutically effective amount of radiolabeled olaratumab or its antigen-binding fragment.

[0056] In any aspect of the present invention, any pharmaceutical or pharmaceutically acceptable composition described herein may be suitable for administration to the abdominal cavity, tumor, topically, or orally, intravenously, or into the airway, preferably by inhalation, or intranasal, subcutaneous, or intramuscular means. Typically, any pharmaceutical or pharmaceutically acceptable composition described herein is suitable for intravenous administration.

[0057] In any method or use described herein, radiolabeled olaratumab or its antigen-binding fragment may be administered as monotherapy for cancer or in combination with one or more additional therapies. Radiolabeled olaratumab or its antigen-binding fragment may be administered after prior treatment for cancer (optionally, prior treatment includes surgery, chemotherapy, immunotherapy, external beam radiation, autologous stem cell therapy, or a combination thereof). Radiolabeled olaratumab or its antigen-binding fragment may be administered concurrently with or sequentially with additional treatment for cancer (optionally, additional treatment includes surgery, chemotherapy, immunotherapy, external beam radiation, autologous stem cell therapy, or a combination thereof).

[0058] Preferably, according to the embodiments and models described above, it will be understood that cancer expresses or overexpresses PDGFR-alpha.

[0059] The cancer may be selected from a list consisting of soft tissue sarcoma (STS), chondrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma. The method of the present invention may enable the treatment, prevention, or minimization of cancer progression characterized by the expression of PDGFR alpha in tumor cells and / or tumor stroma.

[0060] In any embodiment, the cancer may be precancerous or non-metastatic. In any embodiment, the cancer may be malignant or metastatic.

[0061] In any embodiment, the method further includes identifying a subject in need of the treatment described herein. The method may include identifying a subject having cancer that expresses or overexpresses PDGFR-alpha.

[0062] As used herein, unless the context requires otherwise, the term “comprise” and variations such as “comprising,” “comprises,” and “comprised” are not intended to exclude further additives, ingredients, integers, or steps.

[0063] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraph will become apparent from the following description, which is given by example and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0064] [Figure 1] Summary of DFOsq- and DOTA-bioconjugate olaratumab. [Figure 2] Setting up a Biacore surface plasmon resonance assay [Figure 3] Total and surface expression of PDGFRA in KRIB, HuO9, and A-204 sarcoma cell lines. A. Total expression of PDGFRA was determined using Western blotting. Surface expression of PDGFRα on unfixed cells was determined by FACS. [Figure 4] Western blot showing olaratumab-mediated inhibition of the PDGFRA activation pathway. [Figure 5] A) Binding of 89Zr-DFOSq-olaratumab to A204 cells and HCC827 cells at 1 hour; B) Binding of 89Zr-DFOSq-olaratumab and 177Lu-DOTA-olaratumab to A204 cells at 1 hour and 4 hours. [Figure 6] Examples of PET images at 24, 48, and 120 hours are shown. For each image, SUVmax, tumor:background ratio, tumor:liver ratio, and tumor:bone ratio were measured. [Figure 7] In vivo distribution of 89Zr-DFOsq-olaratumab at 24, 48, and 120 hours, measured by radioactivity counts in the blood and dissected lungs, hearts, livers, kidneys, muscles, spleens, bones, and tumors of A204-carrying nude mice. [Figure 8]Mean tumor volume after treatment with radiolabeled olaratumab. Mean tumor volume ± SEM measured until the first mouse from each group reached the tumor endpoint in A204 xenograft tumors in Balb / c nude mice, in both the treatment group (177Lu-olaratumab, 10 MBq) and the control group (cold-DOTA-olaratumab). [Figure 9] This shows the individual tumor volumes from the first day (Day 1) of treatment / vehicle use to the end of the experiment on Day 90. [Figure 10] Survival time measured in the treatment group (177Lu-olaratumab, 10 MBq) and the control group of A204 xenograft tumors in Balb / c nude mice.

[0065] Sequence information [Table 1-1] [Table 1-2] [Modes for carrying out the invention]

[0066] Platelet-derived growth factor receptor alpha (PDGFR alpha) Platelet-derived growth factor receptor alpha (PDGFRα or PDGFR alpha) is a type III receptor tyrosine kinase. PDGFRα is important for development and plays a vital role until adulthood. For example, homozygous mice with a null mutation die during embryonic development. In the later stages of development, PDGFRα is expressed in many mesenchymal structures, while adjacent epithelial cells produce platelet-derived growth factor (PDGF).

[0067] The platelet-derived growth factor family consists of five distinct disulfide-linked dimers, PDGF-AA, -BB, -AB, -CC, and -DD, which act via PDGFRα and PDGFRβ. These growth factors are dimeric molecules composed of disulfide-linked polypeptide chains that simultaneously bind to two receptor proteins, inducing receptor dimerization, autophosphorylation, and intracellular signaling. PDGFRα can form heterodimers with PDGFRβ, as well as homodimers. Because PDGFRβ does not bind to the PDGF-A chain with high affinity, PDGF-AA activates only the αα receptor dimer, while PDGF-AB and PDGF-CC activate both αα and αβ receptor heterodimers.

[0068] PDGFR-alpha has also been detected in several tumors and stromal cells, including sarcomas, suggesting that its signaling may contribute to cancer cell proliferation, metastasis, and the maintenance of the tumor microenvironment.

[0069] Olaratumab (Lartruvo®) is a fully human IgG1 monoclonal antibody that selectively binds to the human platelet-derived growth factor (PDGF) receptor α (PDGFRα). The interaction between olaratumab and PDGFR-α disrupts receptor binding by PDGF-AA and -BB ligands, as well as PDGF-AA, -BB, and -CC-inducible receptor activation, and downstream PDGFR-α pathway signaling. Olaratumab demonstrated antitumor activity against selected sarcoma cell lines in vitro and in vivo, and disrupted the PDGFR-α signaling pathway in a tumor transplantation model in vivo.

[0070] In 2019, Lartruvo® failed to meet the primary overall survival endpoint in a Phase III validation trial (ANNOUNCE trial; NCT02451943) in patients with soft tissue sarcoma. However, the inventors surprisingly found that radiolabeled olaratumab or its antigen-binding fragments may be useful in treating a range of cancers characterized by PDGFRα expression.

[0071] General definition Throughout this Specification, unless otherwise specified or the context requires, references to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions, groups of steps, or groups of compositions of a substance. Accordingly, as used herein, the singular forms "a," "an," and "the" include multiple aspects, and vice versa, unless the context clearly indicates otherwise. For example, a reference to "a" includes not only one but two or more; a reference to "an" includes not only one but two or more; a reference to "the" includes not only one but two or more, and so on.

[0072] Those skilled in the art will understand that the present invention is susceptible to modifications and alterations other than those specifically described. It should be understood that the present invention includes all such modifications and alterations. The present invention also includes, individually or collectively, all of the steps, features, compositions and compounds referred to or indicated herein, as well as any and all combinations, or any two or more such steps or features.

[0073] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described.

[0074] All patents and publications referenced herein are incorporated in their entirety by reference.

[0075] The present invention should not be limited to the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0076] Any example or embodiment of the present invention described herein shall be construed as applying mutatis mutandis to any other example or embodiment of the present invention unless otherwise specified.

[0077] Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as that generally understood by those skilled in the art (for example, in diagnostic techniques, radiographic imaging, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0078] The term "and / or," for example "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as explicitly supporting both meanings or either meaning.

[0079] Main definitions The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, by its origin or the source of its derivatives, does not associate with its naturally associated components in its natural state and substantially does not contain other proteins from the same source. Proteins may be substantially free of naturally associated components by using protein purification techniques known in the art, or substantially purified by isolation. “Substantially purified” means that the protein is substantially free of contaminants, for example, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of contaminants.

[0080] The term "recombinant" shall be understood to mean the product of artificial genetic modification. Therefore, in the context of recombinant proteins containing antibody-antigen-binding domains, this term does not include naturally occurring antibodies within the subject's body, which are products of natural recombination occurring during B cell maturation. However, if such antibodies are isolated, they should be considered isolated proteins containing antibody-antigen-binding domains. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody-antigen-binding domain. Recombinant proteins also include proteins expressed by artificial recombinant means, for example, if they are present within a cell, tissue, or subject.

[0081] The term "protein" shall be interpreted as including a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0082] The terms "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0083] As used herein, the term “antigen-binding protein” is to be used interchangeably with “antigen-binding domain” and is to be interpreted as meaning a region of an antibody capable of specifically binding to an antigen (i.e., VH or VL, or Fv containing both VH and VL). The antigen-binding domain does not need to be in the context of the entire antibody and may be, for example, isolated (e.g., a domain antibody) or in another form, such as those described herein (e.g., scFv).

[0084] For the purposes of this disclosure, the term “antibody” includes proteins that are capable of specifically binding to one or more closely related antigens by an antigen-binding domain contained within the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-transplant antibodies, primate-transplant antibodies, deimmunized antibodies, synthetic humanized antibodies, semi-antibodies, bispecific antibodies). Antibodies generally include a constant domain that can be located in a constant region or a constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50–70 kD) and two light chains (about 23 kDa each). The light chains generally include a variable region (if present) and a constant domain, which in mammals are either κ-light chains or λ-light chains. A heavy chain generally consists of a variable region and one or two constant domains linked to additional constant domains by a hinge region. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently bonded to one of the heavy chains. For example, two heavy chains, as well as a heavy chain and a light chain, 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 to the first constant domain of the heavy chain (CH1, 330-440 amino acids long). The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional CH domains (such as CH2, CH3), and may include a hinge region between the constant domains of CH1 and CH2. 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. For example, the antibody may be a mouse (mouse or rat) antibody or a primate (e.g., human) antibody.In one example, the antibody heavy chain lacks a C-terminal lysine residue. In another example, the antibody is humanized, synthetically humanized, chimeric, CDR-transplanted, or deimmunized.

[0085] 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. Specifically, whole antibodies include those having heavy and light chains containing an Fc region. The constant domain may be the wild-type sequence constant domain (e.g., the human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0086] As used herein, “variable region” refers to a portion of the light and / or heavy chain of an antibody as defined herein, which is capable of specifically binding to an antigen and includes the amino acid sequences of complementarity-determining regions (CDRs), namely CDR1, CDR2, and CDR3, and the framework region (FR). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0087] As used herein, the term “subject” shall be interpreted as meaning any animal, including humans, e.g., mammals. Examples of subjects include, but are not limited to, humans and non-human primates. For example, the subject is humans.

[0088] Antibodies, immunoglobulins, or Igs are gamma globulin proteins found in the blood or other bodily fluids of vertebrates that function in the immune system to bind to antigens, thereby recognizing and neutralizing foreign substances.

[0089] Antibodies are generally heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0090] The heavy and light chains define specific Ig domains. More specifically, each heavy chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain.

[0091] Antibodies can be assigned to different classes or isotypes. Immunoglobulins have five classes: IgA, IgD, IgE, IgG, and IgM, each with heavy chains called α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively slight differences in CH sequences and function. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Light chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains.

[0092] The constant domain contains the Fc region, which includes the carboxyl-terminal portions of both H chains held together by the disulfide. The effector function of antibodies such as ADCC is determined by the sequence of the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0093] The pairing of VH and VL together forms a “variable region” or “variable domain” containing the amino-terminal domains of the antibody’s heavy or light chain. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” The V domain contains the antigen-binding protein, which influences antigen binding and defines the specificity of a particular antibody to its particular antigen. The V region spans approximately 110 amino acid residues and consists of a relatively invariant stretch of 15-30 amino acids (called the framework region (FR) (generally about 4)) separated by a much shorter, highly variable region (called the “hypervariable region” (generally about 3)) with each amino acid length of 9-12. The FR primarily employs a β-sheet configuration, while the hypervariable region forms loops that connect, and sometimes form part of, the β-sheet structure.

[0094] "Hypervariable region," "HVR," or "HV" refers to a region of the antibody's variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Generally, antibodies contain six hypervariable regions, three of which are located in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several methods for defining hypervariable regions have been used and are incorporated herein.

[0095] As used herein, the term “complementarity-determining region” (synonym: CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the antibody variable region whose presence significantly contributes 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 herein as CDR H1, CDR H2, and CDR H3, respectively, with CDR H1 corresponding to CDR1 of VH, CDR H2 to CDR2 of VH, and CDR H3 to CDR3 of VH. Similarly, the CDRs of VL are referred herein as CDR L1, CDR L2, and CDR L3, respectively, with CDR L1 corresponding to CDR1 of VL, CDR L2 to CDR2 of VL, and CDR L3 to CDR3 of VL. In one example, the amino acid positions assigned to CDR and FR are defined according to 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 CDR and FR are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html).The present invention is not limited to FR and CDR as defined by the Kabat numbering system, but includes all numbering systems, including standard numbering systems, namely the numbering systems 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 Plukthun J.Mol.Biol.309:657-670,2001, or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res.25:206-211 1997. For example, CDR is defined according to the Kabat numbering system. Selectively, heavy chain CDR2s assigned to the Kabat numbering system either do not contain the five C-terminal amino acids listed herein, or one or more of these amino acids are substituted with other native amino acids. In this regard, Padlan et al. (FASEB J., 9:133-139, 1995) demonstrated that the five C-terminal amino acids of heavy chain CDR2s are generally not involved in antigen binding.

[0096] A “framework” or “FR” residue is a variable domain residue other than a hypervariable region or CDR residue as defined herein. The FRs of VH are also referred herein as FR H1, FR H2, FR H3, and FR H4, respectively, with FR H1 corresponding to FR1 of VH, FR H2 to FR2 of VH, FR H3 to FR3 of VH, and FR H4 to FR4 of VH. Similarly, the FRs of VL are referred herein as FR L1, FR L2, FR L3, and FR L4, respectively, with FR L1 corresponding to FR1 of VL, FR L2 to FR2 of VL, FR L3 to FR3 of VL, and FR L4 to FR4 of VL.

[0097] "Peptides for forming antigen-binding proteins" generally refer to peptides that can form conformations that confer specificity of antibodies to an antigen. Examples include whole antibodies or whole antibody-related structures, whole antibody fragments including variable domains, variable domains and their fragments including light and heavy chains, or light and heavy chain fragments that include not all but part of the hypervariable region or constant region.

[0098] An "intact" or "whole" antibody comprises an antigen-binding protein, as well as CL and at least heavy chain constant domains (CH1, CH2, and CH3). The constant domains may be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0099] The "total antibody-related structure" includes the multimerized forms of all antibodies.

[0100] "All antibody fragments containing variable domains" include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0101] A Fab fragment consists of the entire light chain (L chain) along with a variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding protein.

[0102] The Fab' fragment differs from the Fab fragment in that it has a small number of additional residues at the carboxyl terminus of the CHI domain containing one or more cysteines from the hinge region of the antibody. Fab'-SH is the herein name for Fab' in which the cysteine ​​residue of the constant domain has a free thiol group.

[0103] The F(ab')2 fragment closely corresponds to the Fab fragment, which is linked by two disulfides with divalent antigen-binding activity and can still crosslink antigens.

[0104] "Fv" is an antibody fragment containing a complete antigen recognition site and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain, which are strongly noncovalently associated.

[0105] In single-stranded Fv(scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a “dimeric” structure similar to that in double-stranded Fv species. The folding of these two domains yields six hypervariable loops (three from the H chain and three from the L chain), which contribute amino acid residues for antigen binding, conferring antigen-binding specificity to the antibody.

[0106] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked together to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, enabling the scFv to form a desired structure for antigen binding.

[0107] A "single variable domain" is half of the Fv (containing only three antigen-specific CDRs), which has the ability to recognize and bind to the antigen, but has lower affinity than the entire binding site.

[0108] A "diabody" refers to an antibody fragment having two antigen-binding sites, the fragment containing a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing an sFv fragment (see previous paragraph) having a short linker (about 5-10 residues) between the VH domain and the VL domain, thereby achieving interchain pairing of the V domain but not intrachain pairing, resulting in a bivalent fragment (i.e., a fragment with two antigen-binding sites).

[0109] Diabodies may be bivalent or bispecific. A bispecific diabody is a heterodimer of two "crossover" sFv fragments, where the VH and VL domains of the two antibodies are located on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0110] "Isolated antibodies" are antibodies identified, separated, and / or recovered from components of their existing environment. Contaminating components are materials that interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0111] "Human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such an antibody in response to an antigenic challenge, but whose endogenous gene locus has been deactivated.

[0112] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the smallest sequence derived from the non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody), and residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired antibody specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, the humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the immunoglobulin constant region of human immunoglobulin.

[0113] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for a few naturally occurring variations that may be present. Monoclonal antibodies are highly specific, targeting a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. Monoclonal antibodies may be prepared by hybridoma or produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells. Monoclonal antibodies may also be isolated from phage antibody libraries.

[0114] The monoclonal antibodies described herein include “chimeric” antibodies (where a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies (insofar as they exhibit the desired biological activity). The chimeric antibodies of interest herein include “primatized” antibodies comprising a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0115] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Generally, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this invention.

[0116] As used herein, the term “binding” in relation to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, antibodies generally recognize and bind to specific protein structures, not proteins. When an antibody binds to epitope “A”, in a reaction involving labeled “A” and a protein, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) reduces the amount of labeled “A” bound to the antibody.

[0117] As used herein, the terms "specifically binds" or "binds specifically" shall be interpreted as meaning that the antigen-binding proteins of the present invention react with or associate with a particular antigen or cell expressing it more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative antigens or cells.

[0118] As used herein, the term “undetectable binding” shall be understood to mean that an antigen-binding protein, e.g., an antibody, binds to a candidate antigen at levels below 10%, 8%, 6%, or 5% above the background. The background may be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the binding level detected in the presence of a negative control antigen. The binding level is detected using a biosensor analysis (e.g., Biacore) in which the antigen-binding protein is immobilized and in contact with the antigen.

[0119] As used herein, the term “not statistically binding” is understood to mean that the binding level of the antigen-binding protein of the present invention to the polypeptide is not statistically significantly higher than the background (e.g., the level of binding signal detected in the absence of the antigen-binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the binding level detected in the presence of a negative control polypeptide). The binding level is detected using biosensor analysis (e.g., Biacore) in which the antigen-binding protein is immobilized and comes into contact with the antigen.

[0120] Affinity-matured antibodies are antibodies that have one or more modifications to one or more HVRs, resulting in improved antibody affinity to an antigen compared to parent antibodies that do not have those modifications. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity to the target antigen. Affinity-matured antibodies are produced by procedures known in the art.

[0121] "ADCC" refers to a process called antibody-dependent cell-mediated cytotoxicity, an immune response in humans primarily mediated by natural killer (NK) cells. In ADCC, FcyRIII on the surface of NK cells recognizes the Fe region of antibodies that bind to antigens presented on the surface of target cells. This activates NK cells, causing them to release perforin and granzymes, leading to lysis and apoptosis of the target cells.

[0122] "CDC" refers to a complex process called complement-dependent cytotoxicity, which can lead to cell death through the action of a cascade of proteins that can act via one of two main pathways.

[0123] "ADCP" refers to a process called antibody-dependent cell-mediated phagocytosis. In this Fe receptor-mediated process, target cells to which antibodies are bound are taken up by phagocytic cells such as macrophages, monocytes, neutrophils, and dendritic cells. Multiple Fc receptors are involved in this process.

[0124] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. A preferred blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0125] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of the polypeptide of interest.

[0126] As used herein, the “Fc region” is a dimer consisting of two polypeptide chains linked by one or more disulfide bonds, each chain containing CH2 and CH3 domains in addition to some or all of the hinge domain. Each polypeptide chain is referred to as the “Fc polypeptide chain.” To distinguish between the two Fe polypeptide chains, one is referred to herein as the “A chain” and the other as the “B chain.” More specifically, the Fc region intended for use with the present invention is the Fc region of IgG, which may be the Fc region of mammalian or human IgG1, IgG2, IgG3, or IgG4. At least two alleles are known within the Fc region of human IgG1.

[0127] As used herein, “Fc-containing protein” refers to a protein comprising an Fc region as described herein and a binding region that binds to a target molecule. The term “Fc-containing protein” encompasses antibodies or Fc fusion proteins that contain an Fc region.

[0128] The terms “to treat” or “treatment” refer to therapeutic treatment whose purpose is to delay (reduce) an undesirable physiological change or impairment. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of the severity of the disease, stabilization (i.e., non-exacerbating) of the disease, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether detectable or undetectable) (whether partial or complete). Treatment may also mean extending survival time compared to the expected survival time if no treatment is received. Treatment does not necessarily result in complete disappearance of the disease or impairment, but it can reduce or minimize complications and side effects of infection and disease or impairment progression.

[0129] The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammal being treated with it.

[0130] antibody This invention relates to the use of radiolabeled olaratumab or its antigen-binding fragments that specifically bind to PDGFRα for the treatment of cancer. As used herein, olaratumab may also be referred to as IMC-3G3 and is an antibody that specifically binds to human PDGFRα.

[0131] As used herein, the terms “specifically binds” or “binds specifically” shall be interpreted as meaning that the agents for use according to the present invention react with or associate with PDGFR alpha or cells expressing it more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative antigens or cells. For example, an antigen-binding protein that binds to PDGFR alpha with substantially higher affinity (e.g., 1.5 times, 2 times, 5 times, 10 times, 20 times, 40 times, 60 times, 80 to 100 times, 150 times, or 200 times) than to other antigens.

[0132] Methods for evaluating binding to proteins (e.g., PDGFR-alpha) are known in the art, for example, as described in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing a drug (e.g., an antibody) and contacting it with a labeled target (antigen in the case of an antibody). After washing to remove nonspecific binding proteins, the amount of label and, consequently, the amount of bound antigen are detected. Of course, the antigen-binding site can be labeled and the antigen immobilized. Panning assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0133] Steady-state region Any antibody and / or its antigen-binding fragment described herein for use in the present invention may include a constant region of the antibody, which includes an antigen-binding fragment of the antibody fused to Fc.

[0134] Sequences of constant regions useful for the production of antibodies or their antigen-binding fragments described herein can be obtained from several different sources. In some examples, the constant region or portion thereof of a protein is derived from a human antibody. The constant region or portion thereof may 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 the human isotype IgG4 or stabilized IgG4 constant region.

[0135] In various embodiments of the present invention, the Fc region of the antibody may include one or more substitutions to alter effector function (including increasing or decreasing effector function) and cyclic half-life. Various examples of such substitutions and modifications are described in Saunders (2019) Front.Immunol.article 1296 (the entire article is incorporated herein by reference).

[0136] In one example, the Fc region of the constant region has a reduced ability to induce effector function compared to, for example, the Fc region of natural or wild-type human IgG1 or IgG3. 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 evaluating the level of effector function of Fc region-containing proteins are known in the art and / or described herein.

[0137] In one example, the Fc region is an IgG4 Fc region (i.e., from the IgG4 constant region), for example, a human IgG4 Fc region. Suitable sequences of IgG4 Fc regions are obvious to those skilled in the art and / or available in publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0138] In one example, the constant region is the stabilized IgG4 constant region. The term “stabilized IgG4 constant region” would be understood to mean an IgG4 constant region modified to reduce Fab arm exchange or the tendency to undergo Fab arm exchange, or the formation of a semi-antibody or the tendency to form a semi-antibody. “Fab arm exchange” refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and attached light chain (half-chain) are exchanged with a heavy-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 spontaneously in vivo and can be induced in vivo by purified blood cells or by reducing agents such as reduced glutathione. “Semi-antibodies” are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0139] In one example, the stabilized IgG4 constant region contains proline at position 241 of the hinge region according to Kabat's system (see 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 (see Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally serine. After the serine substitution for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which confer mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in the inter-heavy-chain disulfide bond. This is generally defined, according to Kabat's numbering system, as extending from Glu226 to Pro243 in human IgG1. Generally, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond at the same position (see, for example, WO2010 / 080538).

[0140] An example of an additional stabilized IgG4 antibody is one in which the arginine at position 409 in the heavy chain constant region of human IgG4 (according to the EU numbering system) is replaced with lysine, threonine, methionine, or leucine (as described, for example, in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain 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 may contain proline (i.e., a CPPC sequence) at position 241 (as described above).

[0141] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulated Fc region.” For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In yet another example, the Fc region is an IgG1 Fc region containing the following changes: one or more of E233P, L234V, L235A and a deletion of G236, and / or the following changes: one or more of 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). Examples of additional non-immunostimulated 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.

[0142] Antibodies with reduced effector function include antibodies having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (as described in U.S. Patent No. 6,737,056, incorporated herein by reference). Such Fc variants include so-called "DANA" Fc variants with substitutions of alanine at residues 265 and 297, and Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581). For example, an antibody variant may include an Fc region with one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, substitutions are L234A and L235A (LALA) (see, for example, WO2012 / 130831). Substitutions may further include substitution of the proline residue at position 329 (e.g., P329G mutation) to invalidate binding to FcR. Furthermore, modifications resulting in alteration (i.e., reduction) of C1q binding and / or complement-dependent cytotoxicity (CDC) may be made in the Fc region, as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J.Immunol. 164:4178-4184 (2000).

[0143] In some embodiments, the Fc region includes mutations to complement (C1q) and / or Fc gamma receptor (FcγR) binding sites. In some embodiments, such mutations can render antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC) impossible for the antibody. An example of a CDC-deficient antibody is an antibody containing substitutions (e.g., K322A) to one or more of Glu318, Lys320, Pro329, Pro331, and Lys322, where the numbering of residues in the Fc region follows the EU index described by Kabat et al.

[0144] In another example, the Fc region is a chimeric Fc region comprising, for example, at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region includes substitutions 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) (as described, for example, in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S and 427F.

[0145] Additional changes The present invention also aims to provide additional modifications to antibody or antigen-binding proteins, including an Fc region or a constant region.

[0146] Neonatal Fc receptors (FcRn) are crucial to the metabolic fate of IgG class antibodies in vivo. FcRn function to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG class antibodies. The interaction between IgG class antibodies and FcRn is pH-dependent and occurs in a 1:2 stoichiometric ratio; that is, 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).

[0147] In certain embodiments of the present invention, an antibody may comprise one or more amino acid substitutions that increase the half-life of a protein. For example, an antibody may comprise an Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region to the neonatal Fc region (FcRn). For example, the Fc region had increased affinity for FcRn at lower pH (e.g., about pH 6.0), promoting Fc / FcRn binding in endosomes. In one example, the Fc region had increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which promotes the re-release of Fc into the bloodstream after cell recycling. These amino acid substitutions are useful in extending the half-life of a protein by reducing its clearance from the blood.

[0148] Exemplary amino acid substitutions include T250Q and / or M428L according to the EU numbering system, or T252A, T254S and T266F, or M252Y, S254T and T256E, or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US2007 / 0135620 or US7083784.

[0149] In further embodiments, the antibody comprises one or more amino acid substitutions that reduce the half-life of the protein. For example, the antibody comprises an Fc region comprising one or more amino acid substitutions that reduce or decrease the affinity of the Fc region to the neonatal Fc region (FcRn).

[0150] Accordingly, the present invention provides an antibody having substitutions in the constant region's CH2 and / or CH3 domains, comprising substitutions in one or more of the residues His310, His435, His436, and Ile253 (Kabat numbering), thereby modifying the FcRn binding affinity and / or serum half-life of the antibody compared to a naturally occurring antibody.

[0151] For example, the amino acid at positions 310 and / or 435 of the antibody may be alanine, glutamic acid, aspartic acid, leucine, isoleucine, arginine, proline, glutamine, methionine, serine, threonine, lysine, asparagine, phenylalanine, tyrosine, tryptophan, cysteine, valine, or glycine.

[0152] Preferably, the residue at position 310 is selected from alanine, glutamic acid, or glutamine, or the amino acid residue 435 from the heavy chain constant region is selected from arginine, glutamine, or alanine. In another preferred embodiment, the antibody has an alanine residue at position 310 and a glutamine residue at position 435.

[0153] In preferred embodiments of the present invention, the binding affinity of the modified antibody to FcRn and / or serum half-life are reduced by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times. In preferred embodiments of the present invention, the binding affinity of the modified antibody to FcRn and / or serum half-life are reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0154] Antibodies may also contain amino acid substitutions in the residues corresponding to Ser228 and Leu235 in the constant heavy chain region (e.g., Ser228Pro and / or Leu235Glu).

[0155] Protein production The production of the antigen-binding protein of the present invention generally requires an expression vector containing a polynucleotide encoding the antigen-binding protein of the present invention. The polynucleotide encoding the antigen-binding protein of the present invention can be obtained by recombinant DNA technology using techniques known in the art, including the techniques described herein, and can be subcloned into a vector for the production of the antigen-binding protein. Many different expression systems are envisioned, including the use of mammalian cells, including human cells, for the production and secretion of the antigen-binding protein. Examples of cells include 293F, CHO, and NSO cell lines.

[0156] Expression vectors comprising a protein-coding sequence and appropriate transcription and translational regulatory signals can be constructed using methods known in the art. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In certain embodiments, a replicable vector is provided having nucleic acids encoding an antigen-binding protein operably linked to a promoter.

[0157] Cells transfected with an expression vector can be cultured by conventional techniques to produce antigen-binding proteins. Therefore, in certain embodiments, a host cell or cell transfectant is provided containing a polynucleotide encoding the antigen-binding protein of the present invention operably linked to a promoter. The promoter may be heterogeneous. Various host expression vector systems may be available, in certain systems the transcription mechanism of the vector system is particularly suited to the host cell. For example, mammalian cells such as Chinese hamster ovary cells (CHO) can be transfected with a vector containing a major intermediate early gene promoter element derived from human cytomegalovirus. Additionally or alternatively, host cells that modulate the expression of the inserted sequence, or modify and process the gene product as needed, including various forms of post-translational modifications, may be used. Examples of mammalian host cells with specific post-translational modification processes include CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O, and HsS78Bst cells.

[0158] Depending on the intended use of the protein molecule, several bacterial expression vectors may be advantageously selected. For example, if a large amount of antigen-binding protein needs to be produced, a vector that produces a high level of fusion protein product that can be easily purified (e.g., the E. coli expression vector pUR278) may be used. The expression product may be produced in the form of a fusion protein with lacZ. Other bacterial vectors include the pIN vector, etc. When expressing an exogenous polypeptide as a fusion protein with glutathione-S-transferase (GST), the pGEX vector may be used. These fusion proteins are generally soluble and can be easily purified from cell lysates by adsorption and binding to a glutathione-agarose affinity matrix, followed by elution in the presence of free glutathione. Thrombin and / or factor Xa protease cleavage sites may be provided to the expression polypeptide, thereby allowing the cloned target gene product to be released from the GST portion.

[0159] Autographa californica nuclear polyhedron disease virus (AcNPV) can be used as a vector for expressing exogenous genes in insect systems, including Spodoptera frugiperda cells. The specific promoter used may depend on where the protein code is inserted into the sequence. For example, the sequence may be cloned separately into the polyhedrin gene and placed under the control of the polyhedrin promoter.

[0160] Virus-based expression systems, such as adenoviruses, can be used in mammalian cells, thereby allowing the desired coding sequence to be ligated to the adenovirus late promoter and tripartite leader sequences. This chimeric gene may then be inserted into the adenovirus genome using in vitro or in vivo recombination. Insertion into region E1 or E3 yields a viable recombinant virus capable of expressing antigen-binding proteins in infected host cells. Specific start signals, including an ATG start codon and adjacent sequences, may be required for efficient translation of the inserted antigen-binding protein coding sequence. Start and translational control signals and codons can be obtained from a variety of sources, both natural and synthetic. Transcriptional enhancer elements and transcriptional terminators may be used to enhance the efficiency of expression in virus-based systems.

[0161] When long-term, high-yield production of recombinant proteins is required, stable expression is preferred. Generally, selection marker genes are used. After transfection, cells are grown in fortified medium for 1-2 days, and then transferred to a medium containing a selection medium that can screen for cells containing the corresponding selection marker (e.g., antibiotic resistance). As a result, cells with stably incorporated plasmids into their chromosomes grow, form foci, and can then be cloned and grown into cell lines. Herpes simplex virus thymidine kinase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes are examples of genes that can be used in tk-, hgprt-, or aprT- cells, respectively, thereby providing an appropriate selection system. The following genes: dhfr (conferring resistance to methotrexate), gpt (conferring resistance to mycophenolic acid), neo (conferring resistance to aminoglycoside G-418), and hygro (conferring resistance to hygromycin) are examples of genes that can be used in anti-metabolite selection systems.

[0162] The antigen-binding proteins of the present invention can be purified by a recombinant expression system, by known methods including ion-exchange chromatography, affinity chromatography (particularly affinity for specific antigen protein A or protein G), and gel filtration column chromatography, by centrifugation, fractional lysis, or by any other standard technique for protein purification. Purification may be facilitated or assisted by providing the antigen-binding proteins in the form of fusion proteins.

[0163] The antigen-binding protein of the present invention can be produced in large quantities by a scalable process. This can be scaled up from a pilot expression system in the laboratory to an analytical-scale bioreactor (typically a 5 L to about 50 L bioreactor) or a production-scale bioreactor (e.g., but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L). A desirable scalable process is one in which aggregation is at a low to undetectable level as measured by HPSEC or rCGE (typically aggregation of 5% by weight or less of the protein to 0.5% by weight or less of the protein). Additionally or alternatively, in the scalable process, fragmentation as measured with respect to the total peak area representing intact antigen-binding protein is preferably at an undetectable level, and therefore it is desirable that intact antigen-binding protein represents at least 80%, and even more preferably 99.5%, or more of the total peak area. In other embodiments, the scalable process of the present invention produces antigen-binding protein with a production efficiency of about 10 mg / L to about 300 mg / L or more.

[0164] Various techniques have been developed for the production of antibody fragments, including the proteolytic digestion of intact antibodies and recombinant expression in host cells. Regarding the latter, as described below, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, and the antibody fragments can be isolated from antibody phage libraries. Fab'-SH fragments can be recovered directly from E. coli and chemically bound to form F(ab')2 fragments. In another approach, F(ab')2 fragments are isolated directly from recombinant host cell cultures.

[0165] In another embodiment, a vector containing the above-described nucleic acid is provided. The vector may be, for example, in the form of a plasmid, cosmid, viral particle, or phage. A suitable nucleic acid sequence can be inserted into the vector by various procedures. Generally, the DNA is inserted into a suitable restriction endonuclease site using techniques known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The construction of a suitable vector containing one or more of these components is done using standard ligation techniques known to those skilled in the art.

[0166] Antigen-binding sites can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides. The heterologous polypeptide may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide. Generally, the signal sequence may be a component of the vector or a portion of the DNA encoding the antigen-binding site to be inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leaders. In the case of yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader, an acid phosphatase leader, or a C. albicans glucoamylase leader. In the case of mammalian cell expression, signal sequences derived from secretory polypeptides of the same or related species, as well as mammalian signal sequences such as viral secretion leaders, may be used to direct protein secretion.

[0167] The polynucleotide sequence encoding the polypeptide component of the antigen-binding protein of the present invention can be obtained using the standard recombination techniques described above. The polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once the polypeptide-encoding sequence is obtained, it is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of a suitable vector depends mainly on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides.

[0168] Generally, plasmid vectors containing replicons and regulatory sequences derived from species compatible with host cells are used in relation to these hosts. Both expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marker sequences that can provide phenotypic selection in transformed cells. Such sequences are well known for various bacteria, yeasts, and viruses. A replication origin derived from plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides an easy means of identifying transformed cells, is suitable for most Gram-negative bacteria; a 2 μm plasmid origin is suitable for yeast; and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages also contain, or can be modified to contain, promoters that can be used by microorganisms for the expression of endogenous proteins.

[0169] In addition, phage vectors containing replicons and regulatory sequences adapted to host microorganisms can be used as transformation vectors in relation to these hosts. For example, a bacteriophage such as λGEM.TM.-11 can be used to create recombinant vectors that can be used to transform receptive host cells such as E. coli LE392.

[0170] The expression vector of the present invention may contain two or more pairs of promoter cistrons (cistrons are DNA segments containing all the information for the production of a single polypeptide). A promoter is a non-translational regulatory sequence located upstream (5' end) of a cistron that regulates its expression. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of a cistron under its control in response to a change in culture conditions (e.g., presence or absence of nutrients or temperature).

[0171] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be operably ligated to cistron DNA encoding a light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both native promoter sequences and many heterologous promoters may be used to direct the amplification and / or expression of a target gene. In some embodiments, heterologous promoters are utilized because they generally allow for greater transcription and higher yield of the target gene compared to native target polypeptide promoters.

[0172] Promoter recognition by various potential host cells is well known. Suitable promoters for use with prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoter. Promoter for use in bacterial systems also includes a Shine-Dalgarno (SD) sequence operably ligated to the DNA encoding the antigen-binding protein of the present invention. However, other promoters that function in bacteria (e.g., promoters of other known bacteria or phages) are equally suitable. Their nucleotide sequences are publicly available, so that those skilled in the art can operably ligate them to cistrons encoding the target light and heavy chains using linkers or adapters to supply any necessary restriction sites.

[0173] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that directs the membrane-based transport of the expressed polypeptide. Generally, the signal sequence may be a component of the vector or a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In the case of a prokaryotic host cell that does not recognize and process the native signal sequence for a heterologous polypeptide, the signal sequence is replaced, for example, by a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp, or the thermostable enterotoxin II (STII) reader, LamB, PhoE, PeIB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0174] In another aspect, the production of immunoglobulins according to the present invention may occur in the cytoplasm of the host cell and therefore does not require the presence of secretory signaling sequences within each cistron. In this regard, the light and heavy chains of the immunoglobulin are expressed, folded, and assembled to form a functional immunoglobulin in the cytoplasm. Certain host strains (e.g., E. coli trxB strain) provide favorable cytoplasmic conditions for disulfide bond formation, thereby enabling proper folding and assembly of the expressed protein subunits.

[0175] The present invention provides an expression system that can adjust the quantitative ratio of expressed polypeptide components to maximize the yield of the secreted and properly assembled antigen-binding protein of the present invention. Such adjustment is achieved at least partially by simultaneously adjusting the translational intensity of the polypeptide components.

[0176] With respect to expression in eukaryotic host cells, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0177] Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is preferably one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For mammalian cell expression, mammalian signal sequences, as well as viral secretion leaders, such as the herpes simplex gD signal, are available.

[0178] Such precursor DNA regions are ligated into the antibody-coding DNA in the reading frame.

[0179] Generally, origin of replication components are not required in mammalian expression vectors. For example, the SV40 origin may typically only be used to include the initial promoter.

[0180] Expression and cloning vectors typically contain selection genes, also known as selection markers. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) compensate for nutritional deficiencies, or (c) supply essential nutrients not available in complex media (e.g., the gene encoding Bacilli's D-alanine racemase).

[0181] One example of a selection scheme involves using drugs to halt the proliferation of host cells. These cells, successfully transformed with heterologous genes, produce proteins that confer drug resistance and thus survive the selection regimen. Examples of such dominant selection use drugs such as neomycin, mycophenolate, and hygromycin.

[0182] Examples of suitable selection markers for mammalian cells include markers that enable the identification of cells capable of taking up nucleic acids encoding antigen-binding proteins such as DHFR or thymidine kinase, metallothionein-I and -II (preferably primate metallothionein genes), adenosine deaminase, and ornithine decarboxylase. Suitable host cells when wild-type DHFR is used are prepared and grown CHO cell lines lacking DHFR activity (e.g., ATCC CRL-9096). For example, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibodies, wild-type DHFR protein, and another selectable marker (e.g., aminoglycoside 3'-phosphotransferase (APH)) can be selected by cell growth in a medium containing a selectable agent against the selectable marker, such as an aminoglycoside antibiotic (e.g., kanamycin, neomycin, or G418).

[0183] Expression vectors and cloning vectors typically contain a promoter operably linked to an antigen-binding protein encoding a nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0184] Eukaryotic genes generally have an AT-rich region located approximately 25–30 base pairs upstream from the transcription start site. Another sequence found 70–80 base pairs upstream from the transcription start site of many genes is the CNCAAT region (where N can be any nucleotide). The 3' end of most eukaryotic genes contains an AATAAA sequence, which may be a signal for adding a poly(A) tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0185] Examples of suitable promoter sequences for use with a yeast host include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, including enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0186] Other yeast promoters that are inducible promoters with further advantages of transcription controlled by growth conditions include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatases, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization.

[0187] The transcription of antigen-binding proteins from vectors in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), as well as promoters derived from heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, and promoters derived from heat shock promoters, provided that such promoters are compatible with the host cell system.

[0188] The transcription of antigen-binding protein-coding DNA by higher eukaryotes can be enhanced by inserting enhancer sequences into vectors. These enhancer sequences include known ones derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers derived from eukaryotic viruses are used. Examples include the SV40 enhancer (bp100-270) after the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer after the origin of replication, and the adenovirus enhancer.

[0189] Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally available from the 5' untranslated region and, occasionally, the 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments into the untranslated portion of mRNA encoding antigen-binding proteins.

[0190] In another embodiment, cells containing the above-described vector or nucleic acid are provided. The nucleic acid molecule or vector may exist in a gene-modified host cell or host as an independent extragenomic molecule, preferably as a molecule that can replicate, or it may be stably incorporated into the host cell or host genome.

[0191] The host cell of the present invention may be any prokaryotic or eukaryotic cell.

[0192] Examples of prokaryotic cells include those commonly used for cloning, such as E. coli or Bacillus subtilis. Furthermore, eukaryotic cells include, for example, fungal cells or animal cells.

[0193] Suitable examples of fungal cells include yeast cells, preferably of the genus Saccharomyces, and most preferably of the species Saccharomyces cerevisiae.

[0194] Examples of animal cells include insect cells, vertebrate cells, preferably mammalian cells, such as HEK293, NSO, CHO, MDCK, U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, and C33A. These host cells, for example, CHO cells, can provide post-translational modifications to the antibody molecule of the present invention, including removal of the leader peptide, folding and assembly of H (heavy chain) and L (light chain), glycosylation of the molecule on the correct side, and secretion of the functional molecule.

[0195] Further suitable cell lines known in the art can be obtained from cell line repositories such as the American Type Culture Collection (ATCC).

[0196] In another embodiment, an animal containing the above-described cells is provided. In a particular embodiment, an animal and its tissues containing a transgene are useful for the production of the antigen-binding protein of the present invention. The introduction of nucleic acid molecules as transgenes into a non-human host and their subsequent expression can be used for the production of antigen-binding proteins; for example, the expression of such transgenes in the milk of a transgenic animal provides a means of obtaining a quantitative amount of antigen-binding protein. A useful transgene in this regard comprises a nucleic acid molecule of the present invention, for example, a coding sequence for the antigen-binding protein described herein, and is operably linked to a promoter and / or enhancer structure derived from a mammary gland-specific gene such as casein or β-lactoglobulin. The animal may be a non-human mammal, most preferably a mouse, rat, sheep, calf, dog, monkey, or ape.

[0197] Radiolabeled antibodies Those skilled in the art will be familiar with standard methods for conjugating detectable portions of radionuclides (radiolabeled objects) or similar materials to antibodies or their antigen-binding fragments.

[0198] As used herein, the term radionuclide may be used interchangeably with the terms radioisotope and radiolabel.

[0199] In any embodiment of the present invention, the radiolabel can be conjugated to oratomab or its antigen-binding fragment directly by binding to one or more amino acid residues in the protein (e.g., halogenation of tyrosine residues) or indirectly by chelating agents, artificial groups, or linkers linked to radioisotopes (i.e., the radiolabel).

[0200] Examples of suitable chelating agents or linkers include 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-NN',N''(N'''-tetraacetic acid, also known as tetraxetane), TCMC (tetraprimary 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-triazabicyclononane-triacetic acid)diamsal (3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), DTPA (pentetic acid or diethylenetriamine-pentaacetic acid), CHX-A''-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclo The chelating agent may be selected from the group consisting of xane-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(desferrioxamine), DFOsq(DFO-squalamide), and HOPO(3,4,3-(LI-1,2-HOPO)), or other chelating agents described herein. Other known chelating moieties include 3p-C-NETA({4 This includes -[2-(biscarboxymethylamino)-5-(4-nitrophenyl)pentyl]-7-carboxymethyl-[1,4,7]triazanonan-1-yl}acetic acid), 5p-C-NETA(2-({1-[4,7-bis(carboxymethyl)-1,4,7-triazanonan-1-yl]-7-(4-nitrophenyl)heptan-2-yl}(carboxymethyl)amino)acetic acid), NOTA(1,4,7-triazacidone-1,4,7-triazacidone-1,4,7-triacetate), and NODA(1,4,7-triazacidone-1,4-diacetic acid).

[0201] Further preferred chelating agents (including, for example, DOTA and DFO) are disclosed in WO2022 / 133537 (incorporated herein by reference).

[0202] In a particular embodiment, olaratumab is a radioactive isotope 124 It can be covalently bound to I. This isotope is a positron emitter that can be bound to antibodies, for example, those described in Larsson et al. (J.Nucl.Med.33(1992),2020-2023) or US5,185,142 (the details of which are incorporated herein by reference).

[0203] In any embodiment, radiolabeling of proteins or antibodies is achieved by covalent iodination, particularly using the iodogen reagent (1,3,4,6-tetrachloro-3a,6a-diphenylglycoryl). Iodogen labeling is a solid-phase oxidation method similar to the chloramine T method, but is generally considered milder because the reaction takes place on the surface of the oxidizing agent, minimizing substrate exposure (Salacinzki, PRP, et al., Anal. Biochem. 117:136 (1981)).

[0204] Chelating agents containing radioactive metals and other halogenated radioisotopes may bind to antibodies via one or more amino acid residues or reactive moieties in a protein / antibody, including but not limited to one or more lysine residues, tyrosine residues, or thiol moieties.

[0205] In another example, an antibody may be conjugated using a bifunctional linker, such as bromoacetyl, thiol, succinimide ester, TFP ester, maleimide, or any amine or thiol modification chemistry known in the art.

[0206] Those skilled in the art will be familiar with standard methods for conjugating chelating agents to antibodies and their derivatives or fragments. In addition, those skilled in the art will be familiar with approaches for selecting relevant chelating agents for pairing with radioactive metals, such as those described, for example, in Chem.Soc.Rev.,2014,43,260 (incorporated herein by reference).

[0207] In any aspect or embodiment, radiolabeled olaratumab may contain any radioisotope suitable for the treatment of tumors or cancerous masses. The radioisotopes may be used in alpha (α), beta (β), or gamma (γ) therapies.

[0208] A suitable example of a radioactive isotope is 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), 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 and radium-224 ( 223 Ra, 224 Ra), rhenium-186 and rhenium-188 ( 186 Re and 188 Re), Samarium-153( 153 Sm), Scandium-47( 47 Sc), Strontium-90 ( 90 Sr), terbium-149 and terbium-161 ( 149 Tb and 161 Tb), and Yttrium-90 ( 90Y) is one example. In a preferred embodiment, the radionuclide conjugated with the antibody or a fragment thereof is actinium-225 or lutetium-177.

[0209] In any embodiment, the radiolabeled olaratumab antibody or its antigen-binding fragment is 89 Zr-, 225 Ac-, or 177 Lu-olaratumab is a radioactive isotope that is directly or indirectly conjugated to the olaratumab antibody.

[0210] Administration and treatment methods of radiolabeled olaratumab Methods for preparing antigen-binding proteins into a suitable form for targeted administration (e.g., pharmaceutical compositions) are known in the art and include, for example, methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the United States Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0211] Radiolabeled olaratumab antibodies are typically administered as a pharmaceutical composition with a pharmaceutically acceptable carrier (e.g., physiological saline) or other aqueous carrier, usually with optional choice, a protein stabilizer (e.g., human serum albumin (HSA)). The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these formulations, the concentration of the antigen-binding protein according to the present invention can be broadly varied and is mainly selected based on fluid volume, viscosity, body weight, etc., according to the selected specific mode of administration and patient needs. 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 may also be used. Liposomes may also be used as carriers. The vehicle may contain small amounts of additives, e.g., buffers and preservatives, to improve isotonicity and chemical stability.

[0212] Administration refers to the physical introduction of a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art, including those described herein. Pharmaceutical compositions may be formulated from the activators of the present invention as described herein for any suitable route of administration.

[0213] Radiolabeled olaratumab is preferably administered intravenously, preferably by infusion or intravenous injection. Infusion of the antibody is preferably carried out over a maximum of about 30 minutes, more preferably over about 15 minutes. Of course, radiolabeled olaratumab can also be administered by subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0214] The terms “therapeutic effective dose” or “effective dose” generally refer to the amount of radiolabeled olaratumab or its antigen-binding fragments described herein that (i) treat a particular disease, condition, or disorder; (ii) reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) delay the onset of one or more symptoms of a particular disease, condition, or disorder. Undesirable effects, such as side effects, may occur along with the desired therapeutic effect. Therefore, practitioners should balance the potential benefits with the potential risks when determining what the appropriate “effective dose” is.

[0215] For example, with respect to the treatment of a tumor, a therapeutically effective amount of the radiolabeled antibody or composition described herein may suppress tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to an untreated subject. Alternatively, the treatment described herein may cause complete regression of the tumor mass. In other embodiments of the present invention, tumor regression can be observed, which lasts for at least about 10 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, or more.

[0216] A therapeutically effective dose of a drug may also include a “preventive” or “prophylactically effective dose,” which is any amount of radiolabeled olaratumab or its antigen-binding fragment administered to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject at risk of cancer recurrence, thereby inhibiting the development or recurrence of cancer. In certain embodiments, the prophylactic effective dose completely prevents the development or recurrence of cancer. To “inhibit” or “prevent” the development or recurrence of cancer means either to reduce the likelihood of cancer development or recurrence, or to completely prevent the development or recurrence of cancer.

[0217] The terms “treatment” or “to treat” include applying or administering the compounds of the present invention to a subject for the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, reducing, modifying, restoring, reducing exacerbation, relieving, improving, or influencing a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility) of a disease or condition. The term “to treat” means any sign of success in treating or alleviating an injury, pathology, or condition, including objective or subjective parameters such as reduction, remission, reduction of exacerbation rate, reduction of disease severity, stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable to the subject, slowing the rate of degeneration or decay, making the final point of degeneration less debilitating, or improving the physical or mental health of the subject.

[0218] As used herein, minimizing or preventing cancer progression means treating a subject to prevent or delay recurrence or metastasis of a tumor, or to prevent the growth of an existing tumor. Minimizing or preventing cancer progression includes preventing or delaying recurrence of cancer, or preventing the growth of an existing tumor, after treatment for cancer. Prevented recurrence includes, for example, recurrence in the tumor bed after surgical resection. Alternatively, recurrence includes metastasis of cancer to another part of the body. As used herein, the terms “preventing recurrence” and “preventing relapse” are interchangeable.

[0219] The present invention also includes methods for preventing the development of cancer in an individual. For example, an individual in need of cancer prevention may be considered to be at risk of developing cancer, but does not yet have a detectable cancer. An individual at risk of developing cancer may be an individual with a family history of cancer, and / or an individual shown to be at high risk or likely to develop cancer through genetic testing or other tests. An individual may have cancer stem cells, but does not yet have a detectable tumor. It will be understood that methods for preventing the development of cancer include methods for delaying the development of cancer in a subject.

[0220] In some embodiments of this disclosure, the cancer to be treated or prevented, or the cancer whose progression should be minimized, is a soft tissue sarcoma, chondrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, or rhabdomyosarcoma. In one embodiment of this disclosure, the cancer is a primary tumor. In another embodiment, the cancer is a metastatic cancer. In yet another embodiment, the cancer has metastasized.

[0221] As used herein, the term “soft tissue sarcoma” or “STS” refers to a type of cancer that develops in the tissues that connect, support, and surround other body structures. This includes fat, muscle, fibrous tissue, blood vessels, nerves, tendons, joint endothelium, or deep skin tissue and / or joint endothelium. They can be found in any part of the body. There are more than 50 subtypes of soft tissue sarcoma. Types of STS include, but are not limited to, angiosarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, liposarcoma, malignant peripheral nerve sheath tumor, myxofibrosarcoma, rhabdomyosarcoma, solitary fibrous tumor, synovial sarcoma, or undifferentiated pleomorphic sarcoma.

[0222] In any aspect or embodiment of this specification, radiolabeled olaratumab may be administered in combination with (i.e., sequentially, simultaneously, or subsequently with) the administration of another treatment for cancer. In any embodiment, the treatment may be any treatment for cancer, optionally selected from surgery, immunotherapy, external beam radiation, chemotherapy, or radioimmunotherapy.

[0223] Chemotherapy agents are chemical agents or drugs that selectively destroy cancer cells and tissues. Chemotherapy agents may include, but are not limited to, taxane compounds, compounds that act via the taxane mechanism, platinum compounds, anthracycline compounds, antimetabolites, epipodophyllotoxin compounds, camptothecin compounds, or any combination thereof.

[0224] In any aspect of the present invention, radiolabeled olaratumab and additional treatments for cancer may be administered simultaneously, or they may be administered sequentially. For example, the additional treatment (e.g., a chemotherapeutic agent) may be administered before the radiolabeled olaratumab, or the radiolabeled olaratumab may be administered before the additional treatment (e.g., a chemotherapeutic agent). Alternatively, treatment with the additional treatment (e.g., a chemotherapeutic agent) and / or radiolabeled olaratumab may be administered alternately.

[0225] The preferred dose of radiolabeled olaratumab or its antigen-binding fragment for use according to the present invention will vary depending on the condition being treated and / or the subject being treated. This is within the scope of the skills of those skilled in the art, and the preferred dose can be determined, for example, by starting with a suboptimal dose and gradually changing the dose to determine the optimal or useful dose. Alternatively, data from cell culture assays or animal studies may be used to determine an appropriate dose for treatment / prevention. The preferred dose is the ED of the active compound with little or no toxicity. 50 The circulating concentration range includes [specific component]. The dose may vary within this range depending on the form of administration and the route of administration used. The therapeutic / prophylactic effective dose can first be estimated from a cell culture assay. The dose is determined in the cell culture, as indicated by IC. 50The formulation may be developed in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration or amount of the compound that achieves semi-maximal inhibition of symptoms). Such information can be used to more accurately determine an effective dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0226] Those skilled in the art will understand that the dosage of a drug for use according to the method of the present invention depends on various factors, including the age, sex, height, and weight of the person to whom the drug is administered, and is drug-dependent.

[0227] Preferably, olaratumab is administered or infused to the subject in a dose of about 1 mg to about 50 mg, preferably about 5 mg to about 20 mg, and more preferably about 10 mg. The specific activity of the radiolabeled antibody is preferably about 15 to about 20 MBq / mg, and more preferably about 18 to about 19 MBq / mg.

[0228] In certain embodiments, radiolabeled olaratumab is administered by slow infusion at a mass dose of approximately 5–20 mg of olaratumab.

[0229] In some embodiments, an effective amount of antibody or its antigen-binding fragment is administered to a patient identified as having cancer at a loading dose of approximately 15 mg / kg, approximately 20 mg / kg, or approximately 25 mg / kg on each of the first 21-day cycle or on each of the first 28-day cycle, followed by administration of a standard dose of antibody or its antigen-binding fragment at approximately 15 mg / kg, approximately 20 mg / kg, or approximately 25 mg / kg on each of the subsequent 21-day cycle and on each of the subsequent 28-day cycle.

[0230] In some embodiments, the radiolabeled olaratumab or its antigen-binding fragment disclosed herein is administered for a minimum period of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or at least about 10 years. In some embodiments, treatment with the radiolabeled olaratumab or its antigen-binding fragment disclosed herein is administered for a maximum period of up to about 18 months, 16 months, 14 months, 12 months, 10 months, 8 months, or 6 months. The treatment period may range from any of these minimum to any of these maximum periods, for example, from 1 to 18 months.

[0231] As used herein, the terms “subject,” “individual,” and “patient” will be understood to be interchangeable. While the present invention is applicable to humans, it is also useful for therapeutic veterinary purposes. The present invention is useful for domesticated or livestock such as cattle, sheep, horses, and poultry, companion animals such as cats and dogs, and zoo animals.

[0232] kit In another embodiment, a kit or product is provided comprising the radiolabeled olaratumab or antigen-binding fragment described above, preferably for use in the methods or uses outlined herein.

[0233] Optionally, the kit may further include labeling or accompanying documentation, including instructions for use.

[0234] A kit or “product” may include a container and labeling or accompanying information on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and blister packs. Containers may be made from a variety of materials, such as glass or plastic. The container may hold a therapeutic composition that is effective in treating a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). The labeling or accompanying information indicates that the therapeutic composition is used to treat a selected condition. In one embodiment, the labeling or accompanying information includes instructions for use.

[0235] The kit may comprise (a) a therapeutic composition and (b) a second container containing a second active ingredient or active component. The kit in this embodiment of the present invention may further include a statement indicating that, using the and other active ingredients, a disorder can be treated or a cancer-related complication can be prevented. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0236] In certain embodiments, the therapeutic composition may be provided in the form of a disposable or reusable device comprising a receptacle for holding the therapeutic composition. In one embodiment, the device is a syringe. The device may hold 1 to 2 mL of the therapeutic composition. The therapeutic composition may be provided in the device either ready for immediate use or requiring mixing or addition of further components.

[0237] It will be understood that the present invention, as disclosed and defined herein, encompasses all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0238] The following examples are intended to illustrate the present invention but are not in any way limiting.

Example

[0239] Example 1: Synthesis and Characterization of Bioconjugates Using the oraratumumab immunoglobulin antibody (e.g., comprising VH described in SEQ ID NO: 4 and VL described in SEQ ID NO: 12), oraratumumab bioconjugates in the form of DOTA-oraratumumab and DFOsq-oraratumumab were generated.

[0240] Using standard methods, bioconjugation of DOTA-oraratumumab and DFOsq-oraratumumab was performed. 30 mg of each conjugate was synthesized and the analytical characterization of the conjugate was carried out.

Table 2

[0241] The DOTA-NHS conjugate showed high monomer purity (>98%) and values of antibody labeling degree (DOL) of 4.4 and 4.3 for DOTA-oraratumumab 1 and DOTA-oraratumumab 2 (generated using two different cell culture methods, respectively) (data not shown).

Table 3

[0242] Size exclusion chromatography (SEC) screening of the DFOsq-oraratumumab conjugate with DFPsq showed high monomer purity (>98%) after conjugation (data not shown).

[0243] The DFOsq conjugate oraratumumab was further evaluated for the characteristics listed in Table 3 below.

Table 4

[0244] Screening of Biacore antibodies and conjugates for binding to PDGFRα The binding of the generated conjugate and olaratumab to PDGFRα was compared (Sino Biological, #10556-H08H, lot #LC15MY0708) using Biacore MCK surface plasmon resonance-based technology (Figure 2). The instrument used was a Biacore T200. Electrophoresis buffer: HBS-P+ buffer containing 1 mg / ml BSA. Tip: Protein A capture sensor tip. Analysis temperature: 25°C. Flow rate: 30 μl / min. Ligand: Purified antibody, approximately 80 RU (10 μl / min). Analyte: Human PDGFRα. Dilution range: 8 points, 3-fold dilution (270 nM to 0.123 nM). Analyte injection time: 240 seconds. Analyte dissociation time: 1200 seconds. Regeneration: Glycine, pH 1.5. Analysis: 1:1 binding (double reference difference correction).

[0245] The results showed that all the antibodies tested bound to PDGFRα with similar affinity (Table 5). [Table 5]

[0246] Example 2: Verification of PDGFRα expression in selected cell lines The selection of cell lines for subsequent testing in xenograft tumor models is based on data published by Lowery et al, 2017, in which the efficacy of olaratumab has been demonstrated in HuO9 (osteosarcoma) and A-204 (embryonic rhabdomyosarcoma) cell lines.

[0247] Figure 3 shows that total PDGFRA expression and surface PDGFRA expression were confirmed in KRIB, HuO9, and A-204 sarcoma cell lines. All three cell lines expressed PDGFRA, which was most abundant in HuO9 cells, although HuO9 cells were found to have a lower proportion of total PDGFRA localized to the plasma membrane.

[0248] PDGF-AA stimulation in all three cell lines induced phosphorylation of AKT on serine 473. This was abolished by pre-incubation with olaratumab (Figure 4).

[0249] Example 3: Development of an in vivo xenograft model The proliferation characteristics of the KRIB and A204 cell lines were tested in a xenograft model of Balb / c nude mice. Both cell lines showed reproducible proliferation and a good "take-rate." The A204 cell clone was selected for subsequent in vivo distribution studies due to its higher radiosensitivity to ionizing radiation.

[0250] Radiolabeling of DFOSq-olaratumab was evaluated using quality control methods, yielding a radiochemical purity of 94%. Surface binding and internalization of the radiolabeled antibody to selected reference cell lines were evaluated using binding assays and immunoreactivity fraction (IRF) assays.

[0251] The activity ratios for cells were based on an initial pilot study, which showed similar saturated binding at 1.25 and 2.5 kBq / 5 million cells at 1 hour, and no binding to negative control cells (specific activity at 1 MBq / 4 μg). Saturated binding (IRF) was found to be approximately 40% in A204 cells and approximately 3% in the negative control strain (HCC827) at 1 hour (Figure 5A). 30–50% of the total binding was found to be internalized after 1 hour (Figure 5, B).

[0252] Example 4: Diagnostic imaging and in vivo distribution studies Balb / c nude mice (N=4) were injected with 5.03 MBq of antibody at a mass dose of 20 μg, and PET images were taken 1, 2, 4, and 6 days after injection (Figure 6). Blockade was achieved by co-injecting 344 μg of unlabeled antibody conjugate (nominal 17-fold excess). Blockade mice were imaged 1 day after injection and re-imaged at later time points, or collected by in vivo distribution. Mice were euthanized, and tissue was collected for in vivo distribution 1, 2, and 4 days after injection (N=4 at each time point) (Figure 7).

[0253] Imaging and in vivo distribution analysis demonstrated that radiolabeled olaratumab targeted tumors with a high tumor-to-background ratio throughout the study period. At 120 hours post-injection, high tumor uptake of approximately 55% ID / g was observed, demonstrating a tumor-to-background ratio of approximately 20.

[0254] Example 5: Efficacy study in a xenograft tumor model Twenty Balb / c nude mice were subcutaneously transplanted with A204 cells into their right flank. Of these, 16 mice were subsequently subjected to a 10 MBq dose. 177 The mice were treated with Lu-DOTA-olaratumab at an antibody mass dose of 40 μg. Four mice received the vehicle (non-radioactively labeled DOTA-olaratumab).

[0255] Tumor volume (Figures 8 and 9) and mouse survival time (Figure 10) were monitored over several weeks.

[0256] The A204 xenotransplant model showed exponential growth with a doubling time of approximately 7 days in the vehicle control group (cold-DOTA-olaratumab). 177 Treatment with Lu-DOTA-olaratumab induced initial stagnation, as well as gradual tumor regression and suppression (Figures 8 and 9). Three mice were still alive at the end of the study at day 90 (Figure 10), two mice reached the tumor endpoint (days 50 and 68), and two mice were euthanized due to weight loss.

[0257] The difference in tumor volume between the groups (change from day 1) was statistically significant at day 20 (P=0.0003, unpaired t-test), and the difference continued to increase thereafter (however, statistical testing was not possible because the mice reached the endpoint).

Claims

1. Olaratumab antibody or olaratumab antibody bioconjugate suitable for radiolabeling with therapeutic radioisotopes.

2. The olaratumab bioconjugate according to claim 1, wherein the bioconjugate comprises an olaratumab antibody conjugated to a chelating group or linker group selected from 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-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, diamsal, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, and HOPO.

3. Conjugated with radioactive isotopes suitable for therapeutic use, Olaratumab antibody or its antigen-binding fragment for treating, preventing, or minimizing the progression of cancer characterized by or associated with PDGFR alpha expression.

4. An olaratumab antibody or its antigen-binding fragment, or antibody bioconjugate according to any one of claims 1 to 3, comprising an antigen-binding domain that competitively inhibits the binding of an antibody comprising VH containing the sequence described in SEQ ID NO: 4 and VL containing the sequence described in SEQ ID NO:

12.

5. The olaratumab antibody or antigen-binding fragment thereof, or antibody bioconjugate according to any one of claims 1 to 4, wherein the antibody or fragment thereof comprises HCDR1, HCDR2, and HCDR3 antigen-binding domains having a variable heavy chain as defined in SEQ ID NO: 4, and LCDR1, LCDR2, and LCDR3 antigen-binding domains having a VL as defined in SEQ ID NO:

12.

6. The antibody or antigen-binding fragment is (i) Complementarity Determination Region (CDR) 1 containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence described in Sequence ID No. 1, and at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, and at least 89% of the sequence set in Sequence ID No.

2. VH, including CDR2 containing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences, and CDR3 containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences to the sequence described in Sequence ID No.

3. (ii) VH containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences to the sequence described in Sequence ID No. 4, (iii) CDR1 containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence is identical to the sequence described in Sequence ID No. 10, at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least VL, including CDR2 containing a sequence identical to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and CDR3 containing a sequence identical to at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence described in Sequence ID No.

11. (iv) A VL containing at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence described in Sequence ID No.

12. (v) VH, containing CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO:

3. (vi) VH containing the sequence described in Sequence ID No. 4, (vii) A VL containing CDR1 containing the sequence described in SEQ ID NO: 9, CDR2 containing the sequence described in SEQ ID NO: 10, and CDR3 containing the sequence described in SEQ ID NO:

11. (viiii) VL containing the sequence described in sequence number 12, (ix) VH, which includes CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 2, and CDR3 containing the sequence described in SEQ ID NO: 3, and VL, which includes CDR1 containing the sequence described in SEQ ID NO: 9, CDR2 containing the sequence described in SEQ ID NO: 10, and CDR3 containing the sequence described in SEQ ID NO: 11, or (x) Olaratumab antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising an antigen-binding domain comprising VH containing the sequence described in SEQ ID NO: 4 and VL containing the sequence described in SEQ ID NO:

12.

7. (i) Framework region (FR) 1 containing or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in Sequence ID No. 6, or F containing or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in Sequence ID No. 6 R2, FR3 containing or consisting of a sequence identical to or consisting of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the sequence described in Sequence ID No. 8, VH, and (ii) A framework region (FR) containing or consisting of a sequence that is at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence described in Sequence ID No.

13.

1. FR2, described in Sequence ID No. 15, which contains or consists of a sequence that is at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence described in Sequence ID No.

14. The sequence contains or consists of at least approximately 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the same sequence as FR3, the sequence described in Sequence ID No. 16, and at least approximately 80% of the same sequence. Olaratumab antibody or antigen-binding fragment thereof according to claim 6, further comprising a VL, which comprises an FR4 having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the same sequence.

8. (i) VH, which includes a framework region (FR) 1 containing or consisting of the sequence described in SEQ ID NO: 5, FR2 containing or consisting of the sequence described in SEQ ID NO: 6, FR3 containing or consisting of the sequence described in SEQ ID NO: 7, and FR4 containing or consisting of the sequence described in SEQ ID NO:

8. (ii) Olaratumab antibody or antigen-binding fragment thereof according to claim 6, further comprising a VL, which includes a framework region (FR) 1 comprising or consisting of the sequence described in SEQ ID NO: 13, an FR2 comprising or consisting of the sequence described in SEQ ID NO: 14, an FR3 comprising or consisting of the sequence described in SEQ ID NO: 15, and an FR4 comprising or consisting of the sequence described in SEQ ID NO:

16.

9. The antibody or its antigen-binding fragment contains at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of the antibody or its antigen-binding fragment, and / or contains at least about 80%, at least 81%, at least 82%, at least at least about 80%, at least 81%, at least 82%, and at least The olaratumab antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a VL containing 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical sequences, wherein there is no sequence diversity between the VH and VL and the sequences of SEQ ID NOs. 4 and 12 in the CDR, and the antigen-binding protein retains the ability to bind to PDGFR alpha.

10. The olaratumab antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or its antigen-binding fragment comprises VH and / or VL, and VH and / or VL comprises substitutions, deletions, or additions of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, or 20 or less amino acid residues, respectively, compared to the amino acid sequence described in SEQ ID NO: 4 or 12, and the amino acid substitutions, deletions, or additions are not present in the CDR, and the antigen-binding protein retains the ability to bind to PDGFR alpha.

11. The antibody or antigen-binding fragment is (i) Single-domain antibody (sdAb), (ii) single chain Fv fragment (scFv), (iii) Dimer scFv (di-scFv), or (iv) an olaratumab antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, in the form of (ii) or (iii) linked to the constant region, Fc, or heavy chain constant domain (CH) 2 and / or CH3 of the antibody.

12. The antibody or its antigen-binding fragment (i) Diabody, (ii) Triabody, (iii) Tetrabody, (iv) Fab, (v)F(ab')2, (vi) Fv, (vii) Bispecific antibodies or other forms of multispecific antibodies, (viiii) Olaratumab or its antigen-binding fragment according to any one of claims 1 to 10, in the form of one of (i) to (vii) linked to the constant region, Fc, or heavy chain constant domain (CH) 2 and / or CH3 of an antibody.

13. The antibody or its antigen-binding fragment is IgG 1 , IgG 2 , IgG 3 , or IgG 4 The olaratumab antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is in the form of an immunoglobulin such as the above.

14. The olaratumab antibody according to claim 13, wherein the olaratumab antibody comprises a heavy chain constant region defined in SEQ ID NO: 17 and / or a light chain constant region defined in SEQ ID NO:

18.

15. The olaratumab antibody according to claim 13 or 14, wherein the olaratumab antibody comprises the heavy chain described in SEQ ID NO: 19 and / or the light chain described in SEQ ID NO:

20.

16. The olaratumab antibody according to any one of claims 13 to 15, wherein the olaratumab antibody comprises one or more amino acid substitutions in the constant region so as to reduce the in vivo half-life of the antibody.

17. The olaratumab antibody according to any one of claims 13 to 16, wherein the antibody comprises a substitution of one or more residues His310, His435, His436, and Ile253 in the constant region of the antibody.

18. The olaratumab antibody according to any one of claims 13 to 17, wherein the antibody comprises an amino acid substitution at position 310 selected from alanine, glutamic acid, or glutamine.

19. The olaratumab antibody according to any one of claims 13 to 18, wherein the antibody comprises an amino acid substitution at position 435 selected from arginine, glutamine, or alanine.

20. The olaratumab antibody according to any one of claims 13 to 19, wherein the antibody comprises an alanine residue at position 310 and a glutamine residue at position 435.

21. The olaratumab antibody according to any one of claims 13 to 20, wherein the antibody comprises an amino acid substitution at residue Lys322, preferably the substitution being K322A.

22. The olaratumab antibody according to any one of claims 16 to 21, wherein the binding affinity of the antibody to FcRn and / or serum half-life of the antibody is reduced by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to an immunoglobulin that does not contain the same substitution, or the binding affinity of the modified antibody to FcRn and / or serum half-life is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

23. The olaratumab antibody or antigen-binding fragment according to any one of claims 3 to 22, wherein the antibody or antigen-binding fragment is conjugated with a radioisotope suitable for the treatment of a tumor or cancerous mass.

24. where the radioisotope is 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), 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 and radium-224 ( 223 Ra, 224 Ra), rhenium-186 and rhenium-188 ( 186 Re and 188 Re), samarium-153 ( 153 Sm), scandium-47 ( 47 Sc), strontium-90 ( 90 Sr), terbium-149 and terbium-161 ( 149 Tb and 161 Tb), and yttrium-90 ( 90 Y), the olaratumab antibody or antigen-binding fragment according to claim 23, selected from.

25. The olaratumab antibody or antigen-binding fragment according to claim 24, wherein the radioactive isotope is actinium-225 or lutetium-177, and the radioactive isotope is directly or indirectly conjugated to the olaratumab antibody.

26. The olaratumab antibody according to any one of claims 23 to 25, wherein the radioactive isotope is directly conjugated to the olaratumab, for example, by halogenation of an amino acid residue.

27. The olaratumab according to any one of claims 23 to 25, wherein the radioactive isotope is indirectly linked to the olaratumab or its antigen-binding fragment, for example, via a chelating agent or other binding site.

28. Olaratumab according to claim 27, wherein the olaratumab is conjugated to a chelate portion 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-NN',N''(N'''-tetraacetic acid), TCMC, DO3A, CB-DO2A, NOTA, diamsal, DTPA, CHX-A''-DTPA, TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, and HOPO, or other chelating agents known to those skilled in the art.

29. The aforementioned antibody 177 Lu-Olaratumab or 177 Lu-labeled oraratumab, preferably the above 177 A radiolabeled olaratumab antibody or antigen-binding fragment thereof according to any one of claims 24 to 28, wherein Lu is conjugated to the olaratumab via a linker.

30. A method for treating, preventing, or minimizing the progression of cancer in a subject, wherein the method is - This includes administering to a subject requiring the use of the radiolabeled olaratumab antibody or its antigen-binding fragment as described in any one of claims 3 to 29, A method for treating, preventing, or minimizing the progression of cancer in the subject.

31. A method for minimizing, reducing, or preventing tumor growth in a target, wherein the method is - This includes administering to a subject requiring the use of the radiolabeled olaratumab antibody or its antigen-binding fragment as described in any one of claims 3 to 29, A method for minimizing, reducing, or preventing tumor growth in the subject.

32. A method for minimizing, reducing, or preventing cancer metastasis in a target, wherein the method is - This includes administering to a subject requiring the use of the radiolabeled olaratumab antibody or its antigen-binding fragment as described in any one of claims 3 to 29, A method for minimizing, reducing, or preventing cancer metastasis in the subject.

33. A method for increasing the survival period of a person suffering from cancer, wherein the method is - This includes administering to a subject requiring the use of the radiolabeled olaratumab antibody or its antigen-binding fragment as described in any one of claims 3 to 29, A method for thereby increasing the survival period of the subject.

34. - To treat, prevent, or minimize the progression of cancer in the target population. - To minimize, reduce, or prevent tumor growth in the target area. - To minimize, reduce, or prevent metastasis in the target, - Use of radiolabeled olaratumab or its antigen-binding fragment according to any one of claims 3 to 29 in the manufacture of a pharmaceutical product for increasing the survival time of a subject with cancer.

35. The method according to any one of claims 30 to 33, or the use according to claim 34, wherein the cancer is selected from the list consisting of soft tissue sarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, and rhabdomyosarcoma.

36. The method according to any one of claims 30 to 33 or 35, wherein the cancer is characterized by the expression of PDGFR alpha in tumor cells and / or tumor stroma.

37. The method or use according to claim 35 or 36, wherein the tumor or cancer is non-metastatic.

38. The method or use of the method or use of claim 35 or 36, wherein the cancer is malignant or metastatic.

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