Reagents and methods for imaging cancers expressing PDGFR alpha

Abstract

The present invention relates to reagents for in vivo imaging and detection of cancer expressing platelet-derived growth factor receptor alpha (PDGFR alpha), as well as methods for imaging cancer and methods for selecting patients for treatment, including methods for using the same.

Description

[Technical Field]

[0001] The present invention relates to reagents for in vivo imaging and detection of cancer expressing platelet-derived growth factor receptor alpha (PDGFR alpha), as well as methods for imaging cancer and methods for selecting patients for treatment, including methods for using the same. [Background technology]

[0002] Medical imaging is often used to aid in the diagnosis and staging of various cancers. Such methods can be advantageous in eliminating or reducing the need for invasive techniques (such as obtaining biopsy samples) to confirm diagnoses, which are not always necessary and can sometimes lead to complications.

[0003] Standard medical imaging techniques cannot successfully detect all cancers. Furthermore, while many imaging techniques can detect tumors, they cannot reliably distinguish between benign and malignant tissue.

[0004] Cancer is a heterogeneous disease with great variability in tumor morphology and physiology. While some conventional histological and clinical features correlate with prognosis, the extensive heterogeneity across the entire morphology of cancer, from the cellular to the histological levels, influences the response to therapy and subsequent patient benefit. Therefore, selectively treating cancer patients who will benefit from specific therapies remains a challenge.

[0005] There is a need for improved methods and compositions for use in in vivo detection and / or imaging of various cancers. There is a need for improved methods and compositions for determining the likelihood that a patient will benefit from a particular cancer treatment.

[0006] Any reference to prior art in this specification does not constitute an acknowledgment or suggestion that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art is understood, considered relevant, and / or can be reasonably expected to be combined with other prior art by those skilled in the art. [Overview of the Initiative]

[0007] The present invention relates to agents and methods for detecting, imaging, and diagnosing cancers expressing platelet-derived growth factor receptor alpha (PDGFR alpha), and to methods for identifying targets for treatment with PDGFR alpha inhibitors.

[0008] In a first aspect, the present invention provides an olaratumab antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment - Detection or imaging diagnosis of PDGFR alpha-expressing tumors in the subject, - Diagnosis of PDGFR alpha-expressing cancer in the target population. -Generation of images of PDGFR alpha-expressing cancers in the target population. - Classification of cancers that are sensitive to treatment with PDGFR alpha inhibitors. -Patient classification or selection for eligibility for cancer therapy with PDGFR alpha inhibitors, or -Conjugated to radioisotopes for monitoring the response to treatment in subjects with PDGFR alpha-expressing cancer or those with PDGFR alpha-expressing cancer.

[0009] It will be understood that, in this specification, an olaratumab antibody or its antigen-binding fragment, in which the antibody or its antigen-binding fragment is conjugated with a radioactive isotope, may also be referred to as a radiolabeled olaratumab antibody or its antigen-binding fragment.

[0010] In a second aspect, the present invention provides an olaratumab antibody or olaratumab antibody bioconjugate suitable for radiolabeling using a diagnostic radioisotope.

[0011] 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 may include olaratumab-TMT(6,6''-bis[N,N'',N'''-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2''-terpyridine), olaratumab-DOTA(1,4,7,10-tetraazacyclododecane-N,N',N''(N'''-tetraacetic acid), olaratumab-TCMC, olaratumab-DO3A, olaratumab-CB-DO2A, or olaratumab-NOTA , selected from olaratumab-diamsal, olaratumab-DTPA, olaratumab-CHX-A''-DTPA, olaratumab-TETE, olaratumab-Te2A, olaratumab-HBED, olaratumab-DFO, olaratumab-DFOsq, olaratumab-DFO-NCS, and olaratumab-HOPO, or olaratumab chelated with any chelating agent disclosed in WO2022 / 133537 or any other chelating agent described herein or known to those skilled in the art.

[0012] The olaratumab antibody or its antigen-binding fragment preferably includes an antigen-binding domain that competitively inhibits the binding of an antibody comprising a VH containing the sequence described in SEQ ID NO: 4 and a VL containing the sequence described in SEQ ID NO: 12.

[0013] In any aspect or embodiment of this specification, the olaratumab antibody or its antigen-binding fragment 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.

[0014] As used herein, the complementarity determining region sequences (CDRs) of the antigen-binding proteins of the present invention can be defined according to the IMGT, Chothia, or Kabat numbering system, or any other CDR numbering system known to those skilled in the art.

[0015] In any aspect or embodiment of the present invention, the olalizumab antibody or an antigen-binding fragment thereof is an antigen-binding domain comprising (i) a complementarity determining region (CDR) 1 comprising a sequence 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 set forth in SEQ ID NO: 1, a CDR2 comprising a sequence 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 �8%, 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 set forth in SEQ ID NO: 2, and a CDR3 comprising a sequence 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 set forth in SEQ ID NO: 3, of VH, (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 comprising a sequence 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 set forth in SEQ ID NO: 12 (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3 (vi) a VH comprising the sequence set forth in SEQ ID NO: 4 (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 9, a CDR2 comprising the sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the sequence set forth in SEQ ID NO: 11 (viii) a VL comprising the sequence set forth in SEQ ID NO: 12 (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the sequence set forth in SEQ ID NO: 3, and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 9, a CDR2 comprising the sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the sequence set forth in SEQ ID NO: 11, or (x) an antigen-binding domain 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.

[0016] The Olaparib antibody or an antigen-binding fragment thereof (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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The variable weight and variable light regions of the antigen-binding domain are selectively linked via a linker.

[0024] 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.

[0025] As defined herein, a linker may be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0026] In any aspect or embodiment of the present invention, radiolabeled 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.

[0027] 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)).

[0028] 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.

[0029] 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.

[0030] 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 a reduced effector function.

[0031] 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).

[0032] 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.

[0033] In a particularly preferred embodiment, olaratumab may include the heavy chain described in SEQ ID NO: 19 and / or the light chain described in SEQ ID NO: 20.

[0034] 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 comprising 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).

[0035] 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.

[0036] 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.

[0037] In further embodiments, the antibody also includes an amino acid substitution at residue Lys322. Preferably, the substitution is K322A.

[0038] In a particularly preferred embodiment, the olaratumab antibody comprises the substitutions K322A, H310A, and H435Q.

[0039] In preferred embodiments, the binding affinity for FcRn and / or the serum half-life of the modified antibody 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 invention, the binding affinity for FcRn and / or the serum half-life of the modified antibody is reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0040] <a In any aspect or embodiment, the antibody may also include amino acid substitutions (e.g., Ser228Pro and / or Leu235Glu) at residues corresponding to Ser228 and Leu235 of the constant heavy chain region.

[0041] In any aspect or embodiment, the radiolabeled olaparib may include any radioisotope suitable for in vivo imaging for the detection of tumors or cancer masses. Examples of suitable radioisotopes include fluorine-18 ( 18 F), gallium-67 and gallium-68 ( 67 Ga and 68 Ga), indium-111 ( 111 In), iodine-123 and iodine-124, technetium-99 ( 99m Tc), terbium-155 and terbium-159 ( 155 Tb and 159 Tb), and zirconium-89 ( 89 Zr).

[0042] 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-N,N',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.

[0043] In another example, radiolabeled olaratumab or its antigen-binding fragments are 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.

[0044] Preferably, the radiolabeled olaratumab antibody or its antigen-binding fragment is 89 Zr-Olaratumab or 89 Zr-labeled oraratumab, preferably 89 Zr is conjugated to olaratumab via a linker (for example, 89 Zr-DFO-Oralatumab, 89 Zr-DFO-NCS-olaratumab, or 89 Zr-DFO-Sq-Olaratumab).

[0045] 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.

[0046] In a second aspect, the present invention relates to a method for in vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer in a subject, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, - A method is provided which includes detecting an antibody or its antigen-binding fragment in a target.

[0047] In a third aspect, the present invention relates to a method for diagnosing PDGFR alpha-expressing cancer in a subject, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, - A method is provided which includes determining the presence or absence of an antibody or its antigen-binding fragment in a target.

[0048] According to a second or third aspect of the present invention, the presence of cancer can be determined by comparing the detection of an antibody or fragment, or the determination of the presence or absence of an antibody or fragment, with the background or standard level, thereby enabling imaging diagnosis or detection of cancer in a subject.

[0049] Alternatively or additionally, detection or presence or absence is compared to a disease classification or disease progression model derived from data from one or more individuals, thereby imaging, detecting, or diagnosing cancer in the subject. The model may be derived from individuals known to have cancer or individuals known not to have cancer (or a combination thereof).

[0050] In certain embodiments, detection or determination may result in data used to compile training data for developing deep learning algorithms to enable artificial intelligence-based self-learning for cancer diagnosis and / or staging. Such methods are commonly known in the art and are described, for example, in WO2020 / 144134 and US11,443,201 (incorporated herein by reference).

[0051] The present invention also relates to a method for generating images of PDGFR alpha-expressing cancer in a subject, wherein this method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject suspected of having cancer, preferably the radiolabeled olaratumab being as described herein, -Detect antibodies or their antigen-binding fragments in the target, This provides a method that includes generating images of cancer.

[0052] The present invention relates to a method for generating images of PDGFR alpha-expressing cancer, wherein this method is -Injecting an effective amount of radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the radiolabeled olaratumab being as described herein, -Detect the antibody or its antigen-binding fragment, This provides a method that includes generating images of cancer.

[0053] The generated cancer images are optionally used to create training data for developing machine learning algorithms.

[0054] In a further embodiment, the present invention relates to a method for classifying cancers that are sensitive to treatment with PDGFR alpha inhibitors, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, -Detect antibodies or their antigen-binding fragments in the target, Therefore, if the detection of an antibody or its fragment is below or equal to the background or threshold level, the cancer is classified as sensitive to treatment with a PDGFR-alpha inhibitor, or The present invention provides a method that includes classifying a cancer as unresponsive to treatment with a PDGFR alpha inhibitor if the detection of an antibody or fragment thereof exceeds a background or threshold level.

[0055] In a further embodiment, the present invention relates to a method for classifying cancers that are sensitive to treatment with PDGFR alpha inhibitors, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, -Detect antibodies or their antigen-binding fragments in the target, This allows us to classify cancers as sensitive to treatment with PDGFR alpha inhibitors based on the output of a deep learning algorithm, and to indicate the presence of PDGFR alpha-expressing cancer cells in the target. This provides a method that, based on the output of a deep learning algorithm, does not classify cancer as sensitive to treatment with PDGFR alpha inhibitors, and indicates the absence of PDGFR alpha-expressing cancer cells in the subject.

[0056] In a further embodiment, the present invention relates to a method for classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, -Detect antibodies or their antigen-binding fragments in the target, Therefore, if the detection of antibodies or their fragments exceeds background or standard levels, the patient is classified / selected for cancer therapy with PDGFR alpha inhibitors. This provides a method that includes not classifying / selecting a patient for cancer therapy with a PDGFR alpha inhibitor if the detection of an antibody or fragment thereof is at or below the background or standard level.

[0057] In a further embodiment, the present invention relates to a method for classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors, wherein the method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a subject requiring it, preferably the radiolabeled olaratumab being as described herein, -Detect antibodies or their antigen-binding fragments in the target, This allows us to classify / select patients for cancer therapy with PDGFR alpha inhibitors based on the output of a deep learning algorithm, and to indicate the presence of PDGFR alpha-expressing cancer cells in the subjects. This provides a method that, based on the output of a deep learning algorithm, does not classify / select patients for cancer therapy with PDGFR alpha inhibitors, and indicates the absence of PDGFR alpha-expressing cancer cells in the subjects.

[0058] In a further embodiment, the present invention relates to a method for monitoring the response to treatment of PDGFR alpha-expressing cancer. Accordingly, the present invention relates to a method for determining the response to treatment of PDGFR alpha-expressing cancer or a subject having PDGFR alpha-expressing cancer, wherein the method is - To administer to subjects who have received or are receiving treatment for PDGFR alpha-expressing cancer, radiolabeled olaratumab antibody or its antigen-binding fragment, preferably radiolabeled olaratumab as described herein, -Detecting antibodies or their antigen-binding fragments in the target, -Compare the amount of antibody or antigen-binding fragment detected in the subject with a reference amount of antibody or antigen-binding fragment detected in the subject before treatment. -If the amount of detected antibody or its antigen-binding fragment is lower than the reference amount, it is determined that the cancer has responded to treatment. -If the amount of antibody or its antigen-binding fragment is greater than or equal to the reference amount, it is determined that the cancer did not respond to treatment. The present invention provides a method that includes determining the response to treatment for PDGFR alpha-expressing cancer or a subject having PDGFR alpha-expressing cancer.

[0059] The treatments received or are receiving by the subject may be any treatment for PDGFR alpha-expressing cancer, including, but not limited to, surgery, chemotherapy, immunotherapy (such as CAR-T therapy), radiotherapy (including external beam radiation or immunoradiotherapy), PDGFR alpha inhibitors, and / or combinations thereof.

[0060] Furthermore, the present invention relates to a method for treating cancer patients, wherein this method is - To administer radiolabeled olaratumab antibody or its antigen-binding fragment to cancer patients suspected of having cancer expressing PDGFR alpha or considered to be at risk of having cancer, preferably the radiolabeled olaratumab is as described herein, -Detecting antibodies or their antigen-binding fragments in patients, - If the detection of antibodies or their fragments exceeds background or standard levels, classify the patient as eligible to receive cancer therapy with PDGFR alpha inhibitors. - If the detection of antibodies or their fragments is at or below the background or standard level, the patient should be classified as ineligible to receive cancer therapy with PDGFR alpha inhibitors. - If a patient is classified as eligible to receive cancer therapy with a PDGFR-alpha inhibitor, the patient should be treated with a PDGFR-alpha inhibitor. - A method is provided which includes deciding not to treat a patient with a PDGFR alpha inhibitor if the patient is classified as not eligible to receive cancer therapy with a PDGFR alpha inhibitor.

[0061] Furthermore, the present invention relates to a method for treating cancer patients, wherein this method is - To administer radiolabeled olaratumab antibody or its antigen-binding fragment to cancer patients suspected of having cancer expressing PDGFR alpha or considered to be at risk of having cancer, preferably the radiolabeled olaratumab is as described herein, -Detecting antibodies or their antigen-binding fragments in patients, - Classifying patients as eligible to receive cancer therapy with PDGFR alpha inhibitors based on the output of a deep learning algorithm derived from data compiled from subjects who responded to treatment with PDGFR alpha inhibitors. - Classifying patients as ineligible for cancer therapy with PDGFR alpha inhibitors based on the output of a deep learning algorithm derived from data compiled from subjects who did not respond to treatment with PDGFR alpha inhibitors. - If a patient is classified as eligible to receive cancer therapy with a PDGFR-alpha inhibitor, the patient should be treated with a PDGFR-alpha inhibitor. - A method is provided which includes deciding not to treat a patient with a PDGFR alpha inhibitor if the patient is classified as not eligible to receive cancer therapy with a PDGFR alpha inhibitor.

[0062] The present invention also relates to a method for treating a cohort of cancer patients, wherein this method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a cohort, preferably the radiolabeled olaratumab being as described herein, -Detecting antibodies or their antigen-binding fragments in a cohort. - If the detection of antibodies or their fragments exceeds background or standard levels, classify the patient as eligible to receive cancer therapy with PDGFR alpha inhibitors. - If the detection of antibodies or their fragments is at or below the background or standard level, the patient should be classified as ineligible to receive cancer therapy with PDGFR alpha inhibitors. - If a patient is classified as eligible to receive cancer therapy with a PDGFR-alpha inhibitor, the patients in the cohort should be treated with a PDGFR-alpha inhibitor. - Provides a method that includes deciding not to treat patients in a cohort with PDGFR alpha inhibitors if the patient is classified as ineligible to receive cancer therapy with PDGFR alpha inhibitors.

[0063] The present invention also relates to a method for treating a cohort of cancer patients, wherein this method is -Administering a radiolabeled olaratumab antibody or its antigen-binding fragment to a cohort, preferably the radiolabeled olaratumab being as described herein, -Detecting antibodies or their antigen-binding fragments in a cohort. - Classifying patients as eligible to receive cancer therapy with PDGFR alpha inhibitors based on the output of a deep learning algorithm derived from data compiled from subjects who responded to treatment with PDGFR alpha inhibitors. - Classifying patients as ineligible for cancer therapy with PDGFR alpha inhibitors based on the output of a deep learning algorithm derived from data compiled from subjects who did not respond to treatment with PDGFR alpha inhibitors. - If a patient is classified as eligible to receive cancer therapy with a PDGFR-alpha inhibitor, the patients in the cohort should be treated with a PDGFR-alpha inhibitor. - Provides a method that includes deciding not to treat patients in a cohort with PDGFR alpha inhibitors if the patient is classified as ineligible to receive cancer therapy with PDGFR alpha inhibitors.

[0064] In any of the aforementioned aspects or embodiments of the present invention, the PDGFR alpha inhibitor may be an olaratumab antibody or its functional antigen-binding fragment. Therefore, according to the aforementioned aspects and embodiments, the present invention is thus -Cancers are classified as being sensitive to treatment with olaratumab antibodies or their functional antigen-binding fragments. -Patients are classified according to their eligibility for cancer therapy with olaratumab antibodies or their functional antigen-binding fragments. - Select the patient for treatment with olaratumab antibody or its functional antigen-binding fragment, or - A method for treating patients or a cohort of cancer patients with olaratumab antibody or its functional antigen-binding fragment, Preferably, the method provides an olaratumab antibody for use in the method described herein, comprising the amino acid sequence defined in Table 1 herein. Most preferably, the olaratumab antibody comprises a heavy chain variable as defined in SEQ ID NO: 4 and a light chain variable as defined in SEQ ID NO: 12.

[0065] The present invention also provides radiolabeled olaratumab antibodies or antigen-binding fragments thereof, or compositions comprising them, for use in the following manner: - In vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer, - Diagnosis of PDGFR alpha-expressing cancer, - To generate images of PDGFR alpha-expressing cancer, - Classifying cancers as being sensitive to treatment with PDGFR-alpha inhibitors, - Classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors. - Treating cancer patients or a cohort of cancer patients with PDGFR-alpha inhibitors. Herein, the method is as described herein, and preferably, the radiolabeled olaratumab is as described herein.

[0066] In another aspect, the present invention provides the use of radiolabeled olaratumab antibodies or their antigen-binding fragments in the preparation of compositions for the following: - In vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer, - Diagnosis of PDGFR alpha-expressing cancer, - To generate images of PDGFR alpha-expressing cancer, - Classifying cancers as being sensitive to treatment with PDGFR-alpha inhibitors, - Classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors. - Treating cancer patients or a cohort of cancer patients with PDGFR-alpha inhibitors. Herein, the method is as described herein, and preferably, the radiolabeled olaratumab is as described herein.

[0067] Furthermore, the present invention provides, when used in the following manner, a radiolabeled olaratumab antibody or its antigen-binding fragment, or a composition containing the same: - In vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer, - Diagnosis of PDGFR alpha-expressing cancer, - To generate images of PDGFR alpha-expressing cancer, - Classifying cancers as being sensitive to treatment with PDGFR-alpha inhibitors, - Classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors. - Treating cancer patients or a cohort of cancer patients with PDGFR-alpha inhibitors. Herein, the method is as described herein, and preferably, the radiolabeled olaratumab is as described herein.

[0068] Furthermore, the present invention provides a kit for use in any of the methods described herein, the kit comprising a radiolabeled olaratumab antibody or its antigen-binding fragment as described herein, and optionally instructions for using it for the following purposes: - In vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer, - Diagnosis of PDGFR alpha-expressing cancer, - To generate images of PDGFR alpha-expressing cancer, - Classifying cancers as being sensitive to treatment with PDGFR-alpha inhibitors, - Classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors. - Treating cancer patients or a cohort of cancer patients with PDGFR alpha inhibitors.

[0069] 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.

[0070] Furthermore, nucleic acids or nucleic acid constructs encoding olaratumab antibodies or antigen-binding fragments thereof, or olaratumab bioconjugates, as described herein are provided.

[0071] Furthermore, a host or host cell containing the nucleic acid or nucleic acid construct of the present invention is provided.

[0072] In any of the above embodiments or models, the method of the present invention may further include concentrating a radiolabeled olaratumab antibody or its antigen-binding fragment at the site and / or tissue of the target where the PDGFR alpha antigen is found, prior to the detection step.

[0073] The detection of radiolabeled olaratumab antibody or its antigen-binding fragment will preferably involve determining or detecting the presence or absence of radiation emitted by a radioisotope conjugated to the antibody. In any embodiment or designation, determining or detecting the presence or absence of radiation includes positron emission tomography (PET), SPECT imaging, or a combination thereof. This method may be further complemented by the use of CT, MRI, or other imaging techniques.

[0074] In any aspect or embodiment, the subject requiring it may be any subject suspected of having or at risk of having PDGFR alpha-expressing cancer. Suspicion of having cancer or consideration of risk may be based on any signs or symptoms associated with cancer, a family history of PDGFR alpha-expressing cancer, or a genotype associated with PDGFR alpha-expressing cancer, or a combination thereof.

[0075] In any aspect or embodiment of the present invention, the cancer to be detected, imaged, diagnosed, classified, or selected may be any cancer expressing PDGFR alpha. Preferably, the cancer is selected from the group consisting of soft tissue sarcoma (STS), chondrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma.

[0076] 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.

[0077] 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]

[0078] [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.

[0079] Sequence information [Table 1-1] [Table 1-2] [Modes for carrying out the invention]

[0080] 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. PDGFR alpha 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).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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. Therefore, there is a need to develop new methods for identifying patients who are sensitive to olaratumab treatment.

[0085] This invention is based on the inventors' discovery that radiolabeled olaratumab or its antigen-binding fragments can be used to recognize PDGFRα expressed on the surface of soft tissue sarcoma (STS) cells, thereby enabling the detection and imaging of such cancer cells, and identifying patients who are most likely to benefit from treatment with PDGFRα inhibitors such as olaratumab.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] All patents and publications referenced herein are incorporated in their entirety by reference.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] 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 of them.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] "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.

[0110] 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.

[0111] 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.

[0112] "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.

[0113] 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.

[0114] The "total antibody-related structure" includes the multimerized forms of all antibodies.

[0115] "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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] "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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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).

[0124] 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.

[0125] "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.

[0126] "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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] "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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] "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.

[0137] "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.

[0138] "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.

[0139] 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.

[0140] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of the polypeptide of interest.

[0141] 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.

[0142] 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.

[0143] 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 disease severity, 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 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.

[0144] 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.

[0145] antibody The present invention relates to the use of radiolabeled olaratumab or its antigen-binding fragment for the detection, imaging, and diagnosis of cancer, and for the selection, classification, and stratification of cancer and cancer patients for the treatment with inhibitors of PDGFRα (in certain embodiments, PDGFRα may be olaratumab or its antigen-binding fragment or derivative). As used herein, olaratumab may also be referred to as IMC-3G3 and is an antibody that specifically binds to human PDGFRα.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] 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).

[0153] 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.

[0154] 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).

[0155] 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).

[0156] 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.

[0157] 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).

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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).

[0162] 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.

[0163] 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.

[0164] In further embodiments, the antibody comprises one or more amino acid substitutions that shorten 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).

[0165] 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.

[0166] 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.

[0167] 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.

[0168] In preferred embodiments of the present invention, the binding affinity to FcRn and / or the serum half-life of the modified antibody 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 to FcRn and / or the serum half-life of the modified antibody are reduced by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.

[0169] 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).

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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).

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] Such precursor DNA regions are ligated into the antibody-coding DNA in the reading frame.

[0194] 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.

[0195] 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).

[0196] 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.

[0197] 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).

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] The host cell of the present invention may be any prokaryotic or eukaryotic cell.

[0207] 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.

[0208] Suitable examples of fungal cells include yeast cells, preferably of the genus Saccharomyces, and most preferably of the species Saccharomyces cerevisiae.

[0209] 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.

[0210] Further suitable cell lines known in the art can be obtained from cell line repositories such as the American Type Culture Collection (ATCC).

[0211] 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.

[0212] Radiolabeled antibodies Those skilled in the art will be familiar with standard methods for conjugating detectable portions of radionuclides (radiolabeled) or similar materials to antibodies or their antigen-binding fragments.

[0213] As used herein, the term radionuclide may be used interchangeably with the terms radioisotope and radiolabel.

[0214] 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).

[0215] 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-N,N',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).

[0216] 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).

[0217] 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)).

[0218] 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.

[0219] 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.

[0220] 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).

[0221] Examples of suitable radioactive labels include fluorine-18( 18 F), Gallium-67 and Gallium-68 ( 67 Ga and 68 Ga), Indium-111( 111 In), iodine-123 and iodine-124, technetium-99 ( 99m Tc), Terbium-155 and Terbium-159 ( 155 Tb and 159 Tb), and zirconium-89( 89 Zr) is one example.

[0222] Administration of radiolabeled olaratumab 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.

[0223] 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.

[0224] In certain embodiments, radiolabeled olaratumab is administered by slow infusion at a mass dose of approximately 5–20 mg of olaratumab.

[0225] Radiolabeled olaratumab antibodies are typically administered as a pharmaceutical composition with a pharmaceutically acceptable carrier (e.g., physiological saline) containing a protein stabilizer (e.g., human serum albumin (HSA)), either optionally or selectively. Radiolabeled olaratumab is preferably administered by infusion.

[0226] 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 applied intraperitoneally or intramuscularly.

[0227] Detection method It will be understood that the method for detecting or imaging radiolabeled olaratumab for use according to the present invention depends on the properties of the detectable portion (radiolabeling) of the antibody.

[0228] The detection step is preferably carried out using PET, SPECT, or any other suitable method. Optionally, the detection method includes PET / SPECT, PET / CT imaging, or PET / MRI scanning.

[0229] Examples of in vivo methods for determining the presence or expression of PDGFRα in tumors include the use of in vivo / partial or whole-body imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT). ImmunoPET and immunoSPECT imaging typically involve the use of PDGFRα-binding molecules, such as olaratumab conjugated to radioisotopes, to enable non-invasive imaging of tissues and tumors expressing PDGFRα.

[0230] The in vivo detection step in the above method may be whole-body imaging or, but is not limited to, local imaging of a specific site such as a site where solid tumor growth is expected or possible.

[0231] In the case of SPECT, radiolabeled olaratumab typically contains a drug that is detectable in the form of a gamma-ray emitting radioisotope (radionic nuclide), usually by injection into the bloodstream. Typically, gamma-ray emitting radioisotopes for use in SPECT are: 99m Tc (technetium), 123 I or131 I (iodine), indium-111 ( 111 In), 67 Ga (gallium), and terbium-155 ( 155 Tb) may be mentioned.

[0232] In any embodiment, the detection method may include positron emission tomography (PET). In such a case, the radiolabeled olalizumab typically contains a drug detectable in the form of a positron-emitting radioisotope (radionuclide), usually by injection into the bloodstream. Examples of radioisotopes used in PET include gallium 68 ( 68 Ga), zirconium-89 ( 89 Zr) and terbium-152 ( 152 Tb) may be mentioned.

[0233] After administration (preferably injection) of the radiolabeled olalizumab, it may be practical to wait for a certain period to allow the drug to accumulate at the site of cancer cells expressing the tumor. Typically, this period will be at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days. Preferably, the time between administration of the drug and detection of the drug (e.g., by PET or other methods described herein), the period is typically within about 10 days, or within about 15 days, or within about 20 days.

[0234] When PET is used for cancer imaging diagnosis or detection, the PET imaging diagnosis can preferably be performed within 7 ± 2 days, particularly 5 ± 2 days after injection of the radiolabeled agent, to obtain optimal imaging diagnosis results including accumulation of the drug at the site where PDGFRα is present.

[0235] The cancer to be detected, imaged, diagnosed, or classified The present invention provides a method for identifying, detecting, or imaging cancer in vivo, and / or classifying such cancer for therapeutic purposes. Such a method is expected to be useful in diagnosing cancers expressing PDGFR alpha, particularly soft tissue sarcomas, and preferably does not require additional invasive techniques (such as biopsy and testing) to confirm the diagnosis.

[0236] Therefore, in preferred embodiments, the methods of the present invention enable the diagnosis of any cancer listed herein without requiring additional invasive diagnostic methods, preferably including biopsy-related methods, as the sole, primary, or main mode of diagnosis for the cancer.

[0237] The method of the present invention is also expected to be useful for staging cancer progression or for the success of cancer treatment. Again, such a method offers the advantage of providing a non-invasive means for evaluating cancer in a subject.

[0238] In further embodiments, the method is a method for identifying cancers that are likely to be sensitive to treatment with olaratumab, or a method for selecting patients for subsequent treatment with PDGFRα inhibitors such as olaratumab.

[0239] As used herein, the term "cancer" refers to malignant proliferation or tumor resulting from uncontrolled cell division. The term "cancer" includes primary tumors and metastatic tumors.

[0240] The subjects of cancer diagnosis, detection, or imaging described herein may be subjects suspected of having cancer or subjects at risk of having cancer. Subjects suspected of having cancer may exhibit one or more symptoms of cancer, have a family history of cancer, or have one or more genetic markers indicating risk or possibility of developing cancer. Subjects considered to be at risk of having cancer may exhibit one or more symptoms of cancer, have a family history of cancer, or have one or more genetic markers indicating risk or possibility of developing cancer.

[0241] PDGFRα-expressing cancers that require diagnosis, detection, imaging, classification, treatment selection, or assessment of treatment success may be selected from the group consisting of soft tissue sarcoma (STS), chondrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, rhabdomyosarcoma, or any other cancer associated with PDGFRα expression. Soft tissue sarcomas originate from tissues such as fat, muscle, nerves, tendons, blood vessels, and lymphatic vessels.

[0242] Tumors can be hormone / androgen-dependent or hormone / androgen-independent and may originate from, for example, the prostate, breast, or lung.

[0243] Primary bone tumors classified or detected according to the present invention include, but are not limited to, osteosarcoma, chondrosarcoma, fibrosarcoma, and angiosarcoma. In particular, malignant secondary (metastatic) tumors are far more common than primary bone tumors. Metastatic bone tumors classified or detected according to the present invention can arise from a variety of causes, the most common of which are prostate cancer, breast cancer, or lung cancer. The cause of metastatic bone cancer is usually revealed from the patient's medical history. Tumors can be osteoblastic or osteolytic. PDGF-dependent bone tumors, as well as "bone marrow"-dependent tumors, can also be classified or detected according to the present invention.

[0244] Cancer imaging or diagnosis is typically evaluated by qualitatively assessing the detection of radiolabeled olaratumab after administration and detection, compared to conventional imaging. Quantitative evaluation may include lesion-specific criteria, including standardized uptake values ​​(SUV) (maximum and mean SUV), lean body mass-adjusted SUV (SUL), metabolic tumor volume (MTV), and tumor-to-background ratio (TBR).

[0245] The tumor-to-background ratio (TBR) is typically defined as the ratio of the lesion's standardized uptake value (maximum SUV) to a reference region SUV (e.g., liver, blood pool). Comparisons are made between the number, size, and other characteristics of lesions detected by standard imaging modalities, including PET scans and high-resolution CT / MRI and other potential imaging modalities, on a patient-by-patient, lesion-by-lesion, and indication-by-indication basis.

[0246] The agreement of drug-specific PET / CT with conventional imaging can be evaluated using qualitative visual analysis of imaging (such as the presence or absence of local drug uptake associated with the tumor, as observed in contrast-enhanced CT, MRI, or FDG PET / CT) to assess the concordance of tumor lesion detection between conventional imaging and conventional imaging. The RECIST 1.1 criteria for conventional imaging can be used as the primary tool for comparing concordance with PET.

[0247] In addition to the above, all tumor lesions visualized by conventional imaging can also be compared with PET imaging results.

[0248] Patient selection / classification and monitoring of responses According to the present invention, patients in whom cancer is detected according to the method of the present invention may be selected or classified for subsequent treatment with a PDGFRα inhibitor or antagonist. The present invention also provides a method for identifying cancers that are likely to be sensitive to treatment with a PDGFRα inhibitor. Accordingly, the present invention also provides a method for identifying patients who would benefit from cancer treatment including a PDGFRα inhibitor, including related treatment methods.

[0249] As used herein, cancer “sensitive” to treatment with a PDGFRα inhibitor is understood to be cancer that is inhibited (i.e., no longer grows) or killed after the patient is administered the PDGFRα inhibitor. Sensitivity to treatment will not necessarily mean complete eradication of the cancer, but rather means cessation or slowing of tumor growth or proliferation, prevention of metastasis, or even tumor lysis / cell death.

[0250] PDGFRα antagonists can be extracellular or intracellular antagonists, and multiple antagonists may be used. Extracellular antagonists include, but are not limited to, proteins or other biological molecules that bind to PDGFRα or one or more of its ligands (e.g., PDGF-AA, -AB, -BB, -CC). In one embodiment, the extracellular antagonist inhibits the binding of PDGFRα to its ligand. In one embodiment, the antagonist is an anti-PDGFRα antibody, e.g., IMC-3G3 (also known as olaratumab). In another embodiment, the binding protein is a soluble ligand-binding fragment of PDGFRα. Intracellular IGF-IR antagonists can be biological molecules, but are usually small molecules. In one embodiment, the intracellular PDGFRα antagonist is AG1296. AG1296 (Calbiochem) is an inhibitor of PDGFβ, PDGFβ, and c-KIT, and also reacts with Flt3. Other small molecules that target PDGFR include STI-571 (imatinib mesylate, Gleevec®, Novartis) and SU11248 (sunitinib malate, SUTENT®, Pfizer).

[0251] Preferably, the inhibitor of PDGFRα neutralizes PDGFRα and, in certain embodiments, may be an anti-PDGFRα antibody. Binding of a ligand (e.g., PDGF-AA, PDGF-AB, PDGF-BB, or PDGF-CC) to the extracellular domain of PDGFRα stimulates receptor dimerization, autophosphorylation, activation of the cytoplasmic tyrosine kinase domain within the receptor, and initiation of several signaling and transactivation pathways involved in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Anti-PDGFRα antibodies typically block ligand binding and / or receptor dimerization, inhibiting one or more of autophosphorylation, tyrosine kinase activation, and signaling. The anti-PDGFRα antibodies of the present invention are specific to the extracellular ligand-binding region of PDGFRα and can prevent ligand binding to PDGFRα. Preferably, such anti-PDGFRα antibodies or fragments thereof bind to PDGFRα with at least the same strength as the native ligand of PDGFRα. Alternatively or additionally, the antibody may be specific to a region of the receptor monomer that would otherwise form a receptor dimer interface. Such an antibody may block dimerization but may or may not block ligand binding to the receptor monomer.

[0252] In one embodiment, an anti-PDGFRα antibody reduces PDGFRα phosphorylation by at least about 75%. In other embodiments, phosphorylation is reduced by at least about 85%, or at least about 90%. In one embodiment, as a result of inhibiting PDGFRα signaling, phosphorylation or downstream signaling pathway components (e.g., Akt, p42 / p44, etc.) are reduced by at least about 40%, at least about 60%, or at least about 80%. Receptor neutralization can be determined using defined ligands (e.g., PDGF-AA, -AB, -BB, -CC), mixtures of such ligands, or preparations such as bone marrow aspirate containing PDGF and other stimulant growth factors.

[0253] Neutralization of PDGFRα typically involves inhibition, reduction, inactivation, and / or disruption of one or more of these activities related to signal transduction. Thus, neutralizing PDGFRα has various effects including inhibition, reduction, inactivation, and / or disruption of growth (proliferation and differentiation), angiogenesis (vascular recruitment, invasion, and metastasis), and cell motility and metastasis (cell adhesion and invasiveness).

[0254] Ex vivo assays as described above can also be used to determine the neutralization of PDGFRα. For example, the inhibition of PDGFRα can be assayed using human SKLMS-1 smooth muscle sarcoma cells (American Type Culture Collection (ATCC), Rockville, Md.; ATCC HTB-88 (trademark)) or U118 glioblastoma cells (ATCC HTB-15 (trademark)) stimulated with PDGF-AA. Inhibition of proliferation can be confirmed using PDGFRα-expressing human tumor cells injected into SCID mice.

[0255] PDGFRα antagonists function by inhibiting signal transduction by PDGFRα expressed on the tumor cells themselves or, alternatively, by inhibiting PDGFRα expressed on surrounding stromal cells that receive paracrine stimulation by PDGF expressed by the tumor cells. Thus, antibodies such as EMC-3G3 (olaratumab) and other PDGFRα antagonists are useful in the treatment of tumors characterized by autocrine and / or paracrine stimulation of PDGFRα.

[0256] The methods of the present invention also find utility in monitoring the response of a subject to cancer treatment. The present invention includes monitoring the effectiveness of treatment for PDGFRα-expressing cancers, and the treatment includes, but is not limited to, administration of any one or more of surgery, chemotherapy, immunotherapy, autologous stem cell transplantation (ASCT), external beam radiation, immunoradiation, inhibitors of PDGFR alpha, or combinations thereof.

[0257] Typically, methods for monitoring the response to treatment include comparing the amount of cancer detectable after or during treatment with the amount of cancer detectable before or at an earlier point in time during treatment. According to the method of the present invention, this typically involves comparing the amount of radiolabeled olaratumab detected in the patient or cancer at different time points.

[0258] In certain embodiments, the detection or determination or imaging diagnostic methods described herein may yield data used to compile training data for developing deep learning algorithms to enable artificial intelligence-based self-learning for cancer diagnosis and / or staging. This includes the use of such artificial intelligence-based methods to compile data from individual patients over the course of therapy to assess the progress of treatment.

[0259] Furthermore, this method may include using a deep learning algorithm to stratify or classify subjects with cancer as likely or unlikely to respond to treatment with PDGFR alpha inhibitors. In certain examples, the training data (or the reference dataset underlying the deep learning algorithm) may include data from one or more individuals that previously responded to treatment with PDGFR alpha inhibitors, and in further examples, the training data (or the reference dataset underlying the deep learning algorithm) may include data from one or more individuals that did not respond to treatment with PDGFR alpha inhibitors. The training data may include data from combinations of patients that had different responses to treatment with PDGFR alpha inhibitors.

[0260] In certain embodiments, the imaging or detection data obtained from the methods described herein may be evaluated by comparing them to a disease classification model generated using machine learning techniques with training data (or a reference dataset underlying a deep learning algorithm) that includes data from one or more individuals that previously responded to treatment with a PDGFR alpha inhibitor, and / or data from one or more individuals that did not respond to treatment with a PDGFR alpha inhibitor.

[0261] Therefore, the AI-based approaches described herein can function as a platform for building a variety of tools and techniques for detecting, assessing, and predicting a patient's cancer status. Furthermore, the AI-based tools described herein can identify various important tissue regions throughout a patient's body and then transfer these identifications (e.g., segmentation maps) from anatomical images to various functional images, so they can be used as building blocks for analytical techniques based on various different imaging diagnostic methods that obtain contrast via various different radiopharmaceuticals.

[0262] The use of AI-based technologies that use machine learning techniques (such as convolutional neural network words) to stratify patients for treatment, monitor disease progression, or evaluate or diagnose cancer based on imaging techniques described herein is generally known in the art and is described, for example, in WO2020 / 144134 and US11,443,201 (which are incorporated herein by reference).

[0263] As used herein, a level or quantity higher than the reference dataset or quantity generally refers to an amount of 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%, at least about 90%, or at least 100% or more.

[0264] As used herein, a level or quantity lower than the reference dataset or quantity generally refers to a quantity that is 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%, at least about 90%, or at least 100% lower than the quantity in the reference dataset.

[0265] The same, higher, or lower levels or amounts generally refer to amounts that differ from the reference dataset by about 5% or less, preferably 10% or less.

[0266] Those skilled in the art will be familiar with methods for determining statically significant differences between detectable levels or amounts of radiolabeled olaratumab for the purpose of making the comparisons necessary for the present invention.

[0267] According to this specification, determining the response to treatment in a subject (or individual deemed to have responded to treatment) with PDGFR alpha-expressing cancer or PDGFR alpha-expressing cancer includes stabilization of the disease or slowing or cessation of disease progression. "Response to treatment" refers to a therapeutic action whose purpose is to delay (mitigate) an undesirable physiological change or impairment. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, 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 an extension of survival compared to the expected survival 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.

[0268] As used herein, a positive response to treatment in an individual includes an increase in the individual's progression-free survival. Alternatively, a positive response to treatment in an individual includes an increase in the individual's overall survival. Therefore, the present invention also finds usefulness in predicting the overall survival or progression-free survival of individuals who receive / need treatment for PDGFRα-expressing cancer.

[0269] As used herein, “overall survival” (OS) refers to the period of time a patient diagnosed with cancer is alive from the date of diagnosis or the date of initiation of treatment. In clinical trials, measuring overall survival is one way to determine how well a new treatment is working.

[0270] "Overall survival" or "OS" is well known to those skilled in the art and refers to the fate of a patient after an event, preferably after the initiation or completion of a treatment regime, despite the possibility that the patient's cause of death may not be directly attributable to the effects of the disease (cancer). In other words, it refers to the prognosis that a patient will not die from PDGFRα-expressing cancer, preferably within at least one year, at least two years, at least three years, at least four years, at least five years, at least ten years, or at least fifteen years.

[0271] As used herein, “progression-free survival” (PFS) refers to the period during and after treatment for a disease such as cancer during which a patient lives with the disease but does not experience a worsening of the condition. In clinical trials, measuring progression-free survival is one way to determine how well a new treatment is working.

[0272] "Responding to a treatment regime" refers to a clinically or biochemically favorable and detectable response to treatment. Typically, a favorable response is survival measured at a later point in time after treatment, for example, one, two, three, or four years after treatment.

[0273] 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.

[0274] Optionally, the kit may further include labeling or accompanying documentation, including instructions for use.

[0275] A kit or “product” may include a container and labeling or accompanying documentation on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and blister packs. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold a diagnostic composition effective for imaging or detection, as described herein, 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). Labeling or accompanying documentation indicates that the composition is used to diagnose or detect a selected condition. In one embodiment, labeling or accompanying documentation includes instructions for use.

[0276] Alternatively or additionally, the kit may further include a second container containing pharmaceutically acceptable buffers such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0277] In certain embodiments, the diagnostic composition may be provided in the form of a disposable or reusable device comprising a receptacle for holding the diagnostic composition. In one embodiment, the device is a syringe. The device may hold 1 to 2 mL of the therapeutic composition. The diagnostic composition may be provided in the device either ready for immediate use or requiring mixing or addition of further components.

[0278] In other embodiments, a kit for use in the diagnostic applications described above is provided. The kit includes: - A container for holding a diagnostic composition in the form of radiolabeled olaratumab or its antigen-binding fragments, - Labels or accompanying documents that include instructions for use.

[0279] The kit may comprise (a) a diagnostic composition and (b) a second container containing a second diagnostic agent or a second label. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, etc.

[0280] 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.

[0281] The following examples are intended to illustrate the present invention, but are not intended to limit it. [Examples]

[0282] Example 1: Synthesis and Characterization of Bioconjugates Oraratumab bioconjugates in the form of DOTA-olaratumab and DFOsq-olaratumab were generated using olaratumab immunoglobulin antibodies containing VH as described in SEQ ID NO: 4 and VL as described in SEQ ID NO: 12.

[0283] Bioconjugation of DOTA-olaratumab and DFOsq-olaratumab was performed using standard methods. 30 mg of each conjugate was synthesized, and the conjugates were analytically characterized. [Table 2]

[0284] DOTA-NHS conjugates demonstrated high monomer purity (>98%) and antibody labeling (DOL) values ​​of 4.4 and 4.3 for DOTA-olaratumab 1 and DOTA-olaratumab 2 (generated using two different cell culture methods, respectively) (data not shown). [Table 3]

[0285] Size exclusion chromatography (SEC) screening of DFOsq-olaratumab conjugates containing DFPsq showed high monomer purity (>98%) after conjugation (data not shown)

[0286] DFOsq conjugate olaratumab was further evaluated based on the characteristics listed in Table 3 below. [Table 4]

[0287] 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).

[0288] The results showed that all the antibodies tested bound to PDGFRα with similar affinity (Table 5). [Table 5]

[0289] 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.

[0290] 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.

[0291] 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).

[0292] 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.

[0293] 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.

[0294] 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).

[0295] 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).

[0296] Imaging studies and in vivo distribution analysis demonstrated that 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.

Claims

1. Olaratumab antibody or olaratumab antibody bioconjugate suitable for radiolabeling with diagnostic 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, olaratumab-TETE, Te2A, HBED, DFO, DFOsq, DFO-NCS, and HOPO.

3. Conjugated with radioactive isotopes suitable for in vivo imaging, - Detection or imaging diagnosis of PDGFR alpha-expressing tumors in the subject, - Diagnosis of PDGFR alpha-expressing cancer in the target population. - Generation of images of PDGFR alpha-expressing cancer in the target, - Classification of cancers that are sensitive to treatment with PDGFR alpha inhibitors, - Classification or selection of patients for eligibility for cancer therapy with PDGFR alpha inhibitors, or Olaratumab antibody or its antigen-binding fragment for monitoring the response to treatment in subjects with PDGFR alpha-expressing cancer or PDGFR alpha-expressing cancer.

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 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, comprising 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 83 at least 80% of the sequence of the antibody or its antigen-binding fragment, and / or at least 80% of the sequence of the antibody or its antigen-binding fragment, and / or at least 80% of the sequence of the antibody or its antigen-binding fragment, and / or at least 8 The olaratumab antibody or antigen-binding fragment according to any one of claims 1 to 8, comprising a VL containing 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 antibody or its antigen-binding fragment 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 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 antibody or its antigen-binding fragment 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 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, (viiii) an olaratumab antibody or antigen-binding fragment thereof 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 the antibody.

13. The antibody or 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 or antigen-binding fragment thereof according to any one of claims 1 to 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 any one of claims 1 to 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, or the antigen-binding fragment thereof.

16. The olaratumab antibody according to any one of claims 3 to 15, wherein the antibody comprises a radioisotope suitable for in vivo imaging diagnostics for the detection of tumors or cancerous masses.

17. The radioactive isotope is fluorine-18 ( 18 F), gallium-67 and gallium-68 ( 67 Ga and 68 Ga), indium-111 ( 111 In), iodine-123 and iodine-124, technetium-99 ( 99m Tc), terbium-155 and terbium-159 ( 155 Tb and 159 Tb), and zirconium-89 ( 89 Zr), the olaratumab antibody according to claim 16.

18. The olaratumab antibody according to claim 16 or 17, wherein the radioactive isotope is directly conjugated to the olaratumab, for example, by halogenation of an amino acid residue.

19. The olaratumab according to claim 16 or 17, 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.

20. Olaratumab according to claim 19, 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-N,N',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.

21. Radiolabeled olaratumab, 89 Zr-olaratumab or 89 Zr-labeled oraratumab, and the above 89 Zr is conjugated to the olaratumab via a linker (for example, 89 Zr-DFO-Olaratumab, 89 Zr-DFO-NCS-olaratumab, or 89 Oralatumab according to claim 20, Zr-DFO-Sq-olaratumab.

22. A method for in vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer in a subject, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. - To detect the antibody or its antigen-binding fragment in the subject, - A method comprising imaging diagnosis or detection of the cancer in the subject.

23. A method for diagnosing PDGFR alpha-expressing cancer in a subject, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. - To determine the presence or absence of the antibody or its antigen-binding fragment in the subject, - A method comprising diagnosing the cancer in the subject thereof.

24. The method according to claim 22 or 23, wherein the presence or absence of the antibody or fragment is determined by comparing the detection of the antibody or fragment, or the presence or absence of the antibody or fragment, with a background or standard level, and thereby the presence of the cancer is determined by imaging diagnosis, detection, or diagnosis of the cancer in the subject.

25. The method according to claim 22 or 23, wherein the detection or presence or absence is compared with a disease classification or disease progression model derived from data from one or more individuals, thereby performing imaging diagnosis or detection or diagnosis of the cancer in the subject.

26. A method for generating images of PDGFR alpha-expressing cancer in a subject, wherein the method is - To administer to a subject suspected of having the aforementioned cancer, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, A method comprising generating an image of the cancer.

27. A method for generating images of PDGFR alpha-expressing cancer, wherein the method is - Injecting an effective amount of radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment, A method comprising generating an image of the cancer.

28. The method according to claim 26 or 27, wherein the generated images of the cancer are used to generate training data for the development of a model of disease progression or disease classification, and optionally the model is a machine learning model based on a neural network.

29. A method for classifying cancers that are sensitive to treatment with PDGFR alpha inhibitors, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, Therefore, if the detection of the antibody or its fragment is below or equal to the background or threshold level, the cancer is classified as sensitive to treatment with a PDGFR alpha inhibitor, or A method comprising classifying the cancer as unresponsive to treatment with a PDGFR alpha inhibitor if the detection of the antibody or a fragment thereof exceeds a background or threshold level.

30. A method for classifying cancers that are sensitive to treatment with PDGFR alpha inhibitors, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, This classifies the cancer as sensitive to treatment with PDGFR-alpha inhibitors based on the output of a deep learning algorithm, and indicates the presence of PDGFR-alpha expressing cancer cells in the subject. A method comprising, based on the output of a deep learning algorithm, not classifying the cancer as sensitive to treatment with a PDGFR-alpha inhibitor, and indicating the absence of PDGFR-alpha expressing cancer cells in the subject.

31. A method for classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, If the detection of the antibody or its fragment exceeds the background or standard level, the patient is classified / selected for cancer therapy with a PDGFR alpha inhibitor. A method comprising, thereby, not classifying / selecting a patient for cancer therapy with a PDGFR alpha inhibitor if the detection of the antibody or fragment thereof is at or below the background or standard level.

32. A method for classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors, wherein the method is - To administer to subjects requiring it, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, This allows for the classification / selection of patients for cancer therapy with PDGFR-alpha inhibitors based on the output of a deep learning algorithm, and indicates the presence of PDGFR-alpha expressing cancer cells in the subjects. A method comprising, thereby, not classifying / selecting the patient for cancer therapy with a PDGFR-alpha inhibitor based on the output of a deep learning algorithm, and indicating the absence of PDGFR-alpha expressing cancer cells in the subject.

33. A method for determining the response to treatment of a subject with PDGFR alpha-expressing cancer or PDGFR alpha-expressing cancer, wherein the method is To administer to subjects who have received or are receiving treatment for PDGFR alpha-expressing cancer, a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment described in any one of claims 3 to 21. -Detect the antibody or its antigen-binding fragment in the subject, - Compare the amount of the antibody or antigen-binding fragment detected in the subject with a reference amount of the antibody or antigen-binding fragment detected in the subject before the treatment. - If the amount of the detected antibody or its antigen-binding fragment is lower than the reference amount, it is determined that the cancer has responded to the treatment. - If the amount of the antibody or its antigen-binding fragment is equal to or greater than the reference amount, it is determined that the cancer did not respond to the treatment. A method comprising determining the response to treatment of a subject with PDGFR alpha-expressing cancer or a subject having PDGFR alpha-expressing cancer.

34. The method according to claim 33, wherein the treatment received or currently received by the subject is selected from surgery, chemotherapy, immunotherapy (such as CAR-T therapy), radiotherapy (including external beam radiation or immunoradiotherapy), PDGFR alpha inhibitors, and / or combinations thereof.

35. The method according to any one of claims 26 to 32 or 34, wherein the inhibitor of PDGFR alpha is an olaratumab antibody or a functional antigen-binding fragment thereof.

36. The method according to claim 35, wherein the olaratumab antibody comprises a heavy chain variable defined in SEQ ID NO: 4 and a light chain variable defined in SEQ ID NO:

12.

37. - In vivo imaging diagnosis or detection of PDGFR alpha-expressing cancer, - Diagnosis of PDGFR alpha-expressing cancer, - To generate images of PDGFR alpha-expressing cancer, - Classifying cancers that are sensitive to treatment with PDGFR alpha inhibitors, or - Use of a radiolabeled olaratumab antibody or its antigen-binding fragment, preferably the olaratumab antibody or its antigen-binding fragment according to any one of claims 3 to 21, in the manufacture of a pharmaceutical product for classifying or selecting patients for eligibility for cancer therapy with PDGFR alpha inhibitors.

38. The method according to any one of claims 22 to 36, wherein the method comprises, prior to the detection step, concentrating the radiolabeled olaratumab antibody or its antigen-binding fragment in the site and / or tissue in the subject where the PDGFR alpha antigen is found.

39. The method according to any one of claims 22 to 36, wherein the detection of the radiolabeled olaratumab antibody or its antigen-binding fragment comprises positron emission tomography (PET), SPECT imaging, or a combination thereof.

40. The method according to any one of claims 22 to 36, or the use according to claim 37, wherein the subject requiring it is suspected of having PDGFR alpha-expressing cancer, or is considered to be at risk of having it.

41. The method according to any one of claims 22 to 36, or the use according to claim 37, wherein the cancer detected, imaged, diagnosed, classified, or selected is selected from the group consisting of soft tissue sarcoma (STS), chondrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma.

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