Growth Hormone Receptor Targeting Polypeptides

Abstract

Aspects of the present disclosure relate to growth hormone receptor (GHR) targeting polypeptides, including antibodies, antibody drug conjugates, antibody fragments, antibody-like molecules, and chimeric receptors. Also disclosed herein are nucleic acids encoding such GHR targeting polypeptides and cells comprising such nucleic acids. Methods for treating acromegaly using GHR targeting polypeptides are described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 311,805, filed February 18, 2022, and U.S. Provisional Patent Application No. 63 / 409,537, filed September 23, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted in XML format and is incorporated herein by reference in its entirety. This XML copy, created on Feb. 13, 2023, is named ARCDP0763WO.xml and is 497,796 bytes in size.

[0003] Aspects of the present invention relate to at least the fields of molecular biology and medicine. [Background technology]

[0004] The growth hormone receptor (GHR) is a master regulator of organismal development and growth. It is a member of the type I cytokine receptor family and is specifically and uniquely activated by growth hormone (GH). Activation of GHR signaling leads to changes in cell metabolism, proliferation, survival, migration, and release of cytokines such as IGF1. Consistent with its biological functions, GHR signaling has been implicated in many human conditions such as developmental delay, cancer, aging, longevity, and disorders such as acromegaly.

[0005] Acromegaly is a rare disease with an estimated prevalence of 4 cases per 100,000 people. 10 In most cases, the condition is caused by a pituitary adenoma, which leads to overproduction of human growth hormone (GH) and insulin-like growth factor 1 (IGF-1). 11Increased levels of these hormones lead to a series of metabolic, respiratory, and cardiovascular comorbidities and physical overgrowth that combine to reduce quality of life and life expectancy. Pegvisomant is a GH variant that has been affinity matured by phage display selection and pegylated at different residues; pegvisomant inhibits the binding of the wild-type hormone to the GHR. 15,16 Pegvisomant has several drawbacks. First, it is injected daily (20-40 mg), which can make it difficult for patients to comply with the medication and reduces their quality of life. Second, some patients develop hepatocellular dysfunction, including high hepatic transaminase levels, in addition to side effects such as lipohypertrophy. 17 , suggesting that not all patients will respond well to the drug. Third, the production of pegvisomant is costly as it requires a second purification step after pegylation.

[0006] There is a need for compositions that target GHR and methods for using such compositions in detecting GHR and treating conditions associated with GHR, such as acromegaly. Summary of the Invention

[0007] To address certain needs in the art, the inventors have generated a variety of GHR binding proteins, including Fabs and antibodies, that compete with human growth hormone binding and reduce GHR downstream signaling. The potent inhibitory antibodies of the present disclosure against GHR provide a new strategy for therapeutic intervention against conditions such as acromegaly, cancer, and longevity. The present disclosure describes novel antibodies and antigen-binding fragments, as well as methods of using these antibodies and fragments. Polypeptides comprising the antigen-binding fragments of the present disclosure and compositions comprising the polypeptides, antibodies, and / or antigen-binding fragments of the present disclosure are also described. Nucleic acids encoding the antibodies or antigen-binding fragments of the present disclosure are also described. The disclosure also relates to a nucleic acid encoding an antibody heavy chain, the nucleic acid having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to any one of SEQ ID NOs:415-445. Also described are nucleic acids encoding an antibody light chain of the present disclosure, the nucleic acids having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to any one of SEQ ID NOs: 415-445. Also described are vectors or expression vectors comprising the nucleic acids of the present disclosure, and host cells comprising the polypeptides, nucleic acids, vectors, antibodies, or antigen-binding fragments of the present disclosure. The nucleic acids of the present disclosure may be DNA or RNA.

[0008] Also described are methods of making a cell, comprising introducing one or more nucleic acids of the present disclosure into the cell. The method may comprise or further comprise culturing the cell under conditions that allow expression of a polypeptide from the nucleic acid. The method may further comprise isolating the expressed polypeptide. The cell may be further defined as a human cell, a B cell, a T cell, a Chinese hamster ovary, an NS0 mouse myeloma cell, a PER.C6 cell, or a cell described herein.

[0009] The present disclosure describes a method for treating or preventing acromegaly in a subject, comprising administering to the subject an antibody, antigen-binding fragment, polypeptide, nucleic acid, or host cell of the present disclosure. Also described is a method for evaluating a sample from a subject, comprising contacting a biological sample from the subject or an extract thereof with at least one antibody, antigen-binding fragment, polypeptide, composition, or host cell of the present disclosure. The antibody, antigen-binding fragment, or composition of the present disclosure may be used to treat a subject with acromegaly.

[0010] The methods include a method for treating or preventing cancer in a subject, comprising administering to the subject an antibody, antigen-binding fragment, polypeptide, nucleic acid, or host cell of the present disclosure. Also described is a method for evaluating a sample from a subject, comprising contacting a biological sample from the subject or an extract thereof with at least one antibody, antigen-binding fragment, or polypeptide, composition, or host cell of the present disclosure. Also disclosed is a method for diagnosing cancer in a subject, comprising contacting a biological sample from the subject or an extract thereof with at least one antibody, antigen-binding fragment, composition, or polypeptide of any one of the present disclosure. The antibody, antigen-binding fragment, or composition of the present disclosure may be used to treat a subject with cancer.

[0011] The present disclosure provides an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region of an antibody clone in Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 derived from the light chain variable region of the same clone in Table 1.The present disclosure also relates to an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has 80% or at least 80% sequence identity with HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of an antibody clone in Table 1, or , 95, 96, 97, 98, 99, or 100% (or any range derivable therein) or at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) of sequence identity with the HCDR1, HCDR2. , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or more) sequence identity with LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone in Table 1. 9, 98, 99, 100%, or 110% (or any range derivable therein) of sequence identity to the HCDR1, HCDR2, and LCDR3. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 may be determined from the variable region sequence by methods known in the art.The CDRs may be HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as determined by the Chothia method. The CDRs may be HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as determined by the Kabat method. The CDRs may be HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as determined by the IMGT method.

[0012] The present disclosure provides an antibody or antigen-binding fragment, wherein HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 80% sequence identity with HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, and wherein HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are derived from the same antibody clone. HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 may each comprise an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 in Table 1, and HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are derived from the same antibody clone. HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences of HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 in Table 1, and HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are derived from the same antibody clone.

[0013] The present disclosure relates to an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has HCDR1, HCDR2, and HCR3 that have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the HCDR1, HCR2, and HCR3 from the heavy chain variable region of an antibody clone in Table 1. and a light chain variable region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the light chain variable region of the same antibody clone of Table 1. The antibody or antigen-binding fragment may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 that have 80% or at least 80% sequence identity with HCDR1, HCR2, HCR3 from the heavy chain variable region of an antibody clone in Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 that have 80% or at least 80% sequence identity with LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone in Table 1. In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 that have the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the clone in Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 that have the amino acid sequences of LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same clone in Table 1.

[0014] A polypeptide of the present disclosure may comprise at least two antigen-binding fragments, each of which is independently selected from the antigen-binding fragments of the present disclosure. A polypeptide may be multivalent. A polypeptide may be multispecific. A polypeptide may be bispecific. A polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-binding regions, or may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-binding regions, or may comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-binding regions. Each antigen-binding region may be independently selected from the antigen-binding regions of the present disclosure. A polypeptide may have repeat units of the same antigen-binding region, for example, at least, up to, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units.

[0015] The heavy chain variable region may comprise an amino acid sequence having at least 80% sequence identity to a heavy chain variable region of an antibody clone of Table 1, and / or the light chain variable region may comprise an amino acid sequence having at least 80% sequence identity to a light chain variable region of the same antibody clone of Table 1. The heavy chain variable region may comprise an amino acid sequence having at least 80% sequence identity to a light chain variable region of the same antibody clone of Table 1. The heavy chain variable region may have an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) or less than 60% sequence identity to a heavy chain variable region of an antibody clone of Table 1. and / or an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the or the light chain variable region is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) of the light chain variable region of the same antibody clone in Table 1; may comprise an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the heavy chain variable region of an antibody clone of Table 1, and / or the light chain variable region may comprise the amino acid sequence of the same antibody clone of Table 1.The antibody or antigen-binding fragment comprises heavy chain framework regions (HFR) 1, HFR2, HFR3, and HFR4, and light chain framework regions (LFR) 1, LFR2, LFR3, and LFR4, where HFR1, HFR2, HFR3, and HFR4 comprise amino acid sequences having at least 80% sequence identity with HFR1, HFR2, HFR3, and HFR4, respectively, of the antibody clone in Table 1, and LFR1, LFR2, LFR3, and LFR4 comprise amino acid sequences having at least 80% sequence identity with LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone in Table 1. The antibody or antigen-binding fragment may comprise heavy chain framework regions (HFR) 1, HFR2, HFR3, and HFR4 and light chain framework regions (LFR) 1, LFR2, LFR3, and LFR4, where HFR1, HFR2, HFR3, and HFR4 are at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of the HFR1, HFR2, HFR3, and HFR4 of the antibody clones of Table 1, respectively. % (or any range derivable therein) sequence identity to LFR1, LFR2, LFR3, and LFR4 of the same antibody clone in Table 1, respectively, and LFR1, LFR2, LFR3, and LFR4 may comprise amino acid sequences that have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to LFR1, LFR2, LFR3, and LFR4 of the same antibody clone in Table 1, respectively. HFR1, HFR2, HFR3, and HFR4 may respectively comprise the amino acid sequences of HFR1, HFR2, HFR3, and HFR4 of the antibody clone in Table 1, and LFR1, LFR2, LFR3, and LFR4 may respectively comprise the amino acid sequences of LFR1, LFR2, LFR3, and LFR4 of the same antibody clone in Table 1.The antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70% sequence identity to a heavy chain of an antibody clone in Table 1, and the light chain comprises an amino acid sequence having at least 70% sequence identity to a light chain of the same antibody clone in Table 1. The antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any combination thereof) identical to the heavy chain of the antibody clone in Table 1. , 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the and the light chain comprises an amino acid sequence that is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) of the light chain of the same antibody clone in Table 1. ), or an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the antibody or antigen-binding fragment. The antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of the antibody clone of Table 1 and the light chain comprises the amino acid sequence of the same antibody clone of Table 1.

[0016] The heavy chain variable region may comprise a heavy chain framework region having 80% or at least 80% sequence identity to the heavy chain framework region of the antibody clone of Table 1, and the light chain variable region comprises a light chain framework region having 80% or at least 80% sequence identity to the light chain framework region of the same antibody clone of Table 1. The heavy chain variable region may comprise a heavy chain framework region having 80% or at least 80% sequence identity to the light chain framework region of the same antibody clone of Table 1. The heavy chain variable region may comprise a light chain framework region having 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) or less than 60% sequence identity to the heavy chain framework region of the antibody clone of Table 1. and a light chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the light chain. The chain variable region is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) of the light chain framework region of the same antibody clone of Table 1, or In some embodiments, the light chain framework region has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to the light chain framework region.

[0017] The heavy chain variable region may comprise at least 70% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 70% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 75% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 75% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 80% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 80% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 85% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 85% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 90% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 90% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1. The heavy chain variable region may comprise at least 95% sequence identity with the heavy chain variable region of the antibody clone of Table 1, and the light chain variable region comprises at least 95% sequence identity with the light chain variable region of the same antibody clone of Table 1, and the heavy and light chains comprise 100% sequence identity with each of the three heavy chain CDRs and the three light chain CDRs from the same antibody clone of Table 1.

[0018] The antigen-binding fragment may be at least 2, 3, 4, 5, or 6 scFv, F(ab')2, Fab', Fab, Fv, or rIgG, or a combination thereof. The polypeptides and / or antigen-binding fragments of the present disclosure may include a linker between the heavy and light chain variable regions or between the antigen-binding fragments. The linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS-SEQ ID NO:446)n, (GS)n, and (GGGS-SEQ ID NO:447)n, where n is an integer of at least 1. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art may be used as linkers in the polypeptides of the present disclosure. Exemplary linkers may include or consist of GGSG (SEQ ID NO:448), GGSGG (SEQ ID NO:449), GSGSG (SEQ ID NO:450), GSGGG (SEQ ID NO:451), GGGSG (SEQ ID NO:452), GSSSG (SEQ ID NO:453), and the like.

[0019] The antibodies or antigen-binding fragments of the present disclosure may be human, chimeric, or humanized. -6 M / L or M~approx. 10 -12 M / L or M K D The antibody or antigen-binding fragment may bind to GHR at about 10, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10-17 , or 10 -18 (or any derivable range therein), at least 10, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10 -17 , or 10 -18 (or any derivable range therein), or up to 10, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10 -17 , or 10 -18 (or any derivable range therein) K in M, M / L, μM, nM, or pM D The antibody or antigen-binding fragment may bind to GHR at a K of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, or any derivable range or value therein. D The antibody or antigen-binding fragment may bind to GHR with a K of less than 2 nM. D The antibody or antigen-binding fragment may bind to GHR with a K of less than 1 nM. D The antibody or antigen-binding fragment may bind to GHR with a K of less than 0.5 nM. DThe antibody or antigen-binding fragment may be further defined as a human antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, a recombinant antibody or antigen-binding fragment, a chimeric antibody or antigen-binding fragment, an antibody or antigen-binding fragment derivative, a veneered antibody or antigen-binding fragment, a diabody, a monoclonal antibody or antigen-binding fragment, a single domain antibody, or a single chain antibody. The antigen-binding fragment may be defined as a single chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, or rIgG. The antibody, antigen-binding fragment, or polypeptide may be operably linked to a detectable label. Detectable labels are described herein.

[0020] Multispecific antibodies and polypeptides are also described herein. Thus, bivalent or bispecific antibodies are provided herein that comprise two antigen-binding fragments, where the antigen-binding fragments are two identical or two different antigen-binding fragments. The present disclosure also provides multispecific polypeptides. Polypeptides are also described that comprise at least 2, 3, 4, 5, or 6 antigen-binding fragments. The antigen-binding fragments may be at least 2, 3, 4, 5, or 6 scFv, F(ab')2, Fab', Fab, Fv, or rIgG, or a combination thereof.

[0021] A composition may comprise two or more antibodies and / or antigen-binding fragments of the disclosure. Thus, a composition of the disclosure may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antibodies and / or antigen-binding fragments of the disclosure.

[0022] In embodiments of the present disclosure, it is also contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of any of SEQ ID NOs: 415 to 445 may be excluded.

[0023] The subject may be further defined as a human subject or a mammalian subject. The subject may be a laboratory or veterinary animal, such as a pig, horse, cat, cow, rabbit, mouse, rat, or dog. The subject may have one or more symptoms of cancer. The subject may not have symptoms of cancer. The subject may be a subject diagnosed with cancer. The subject may be a subject not diagnosed with cancer. The subject may be a subject who has previously been treated for cancer. The subject may have one or more symptoms of acromegaly. The subject may not have one or more symptoms of acromegaly. The subject may be a subject diagnosed with acromegaly. The subject may be a subject not diagnosed with acromegaly. The subject may be a subject who has previously been treated for acromegaly. The subject may be administered an additional treatment. The subject may be a subject who is resistant or determined to be resistant to a previous treatment. The additional treatment may include one or more of radiation therapy, chemotherapy, and immunotherapy.

[0024] The method may further include incubating the antibody, antigen-binding fragment, or polypeptide under conditions that allow the antibody, antigen-binding fragment, or polypeptide to bind to the antigen in the biological sample or an extract thereof. The method may further include detecting binding of the antigen to the antibody, antigen-binding fragment, or polypeptide. The method may further include contacting the biological sample with at least one capture antibody, antigen, or polypeptide. The at least one capture antibody, antigen-binding fragment, or polypeptide may be an antibody, polypeptide, or antigen-binding fragment of the present disclosure. The capture antibody may be linked or operably linked to a solid support. The term "operably linked" refers to a situation in which two components can be linked or combined to form a complex. For example, the components may be covalently linked and / or on the same polypeptide, such as in a fusion protein, or the components may have a certain degree of binding affinity for each other, e.g., binding affinity caused by van der Waals forces. The biological sample may include a blood sample, a urine sample, a fecal sample, or a nasopharyngeal sample. At least one antibody, antigen-binding fragment, or polypeptide may be operably linked to a detectable label. The method may further comprise incubating the antibody, antigen-binding fragment, or polypeptide under conditions that allow the antibody, antigen-binding fragment, or polypeptide to bind to the antigen in the biological sample or an extract thereof. The method may further comprise detecting binding of the antigen to the antibody, antigen-binding fragment, or polypeptide. The method may further comprise contacting the biological sample with at least one capture antibody, antigen, or polypeptide. The biological sample may include a blood sample, a urine sample, a fecal sample, or a nasopharyngeal sample.

[0025] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0026] An embodiment of the present disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 14, respectively.

[0027] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 12, and 20, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 21, respectively.

[0028] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 29, respectively.

[0029] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 34, and 35, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 36, respectively.

[0030] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 44, respectively.

[0031] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 52, respectively.

[0032] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 60, respectively.

[0033] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 65, and 66, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 67, respectively.

[0034] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 72, 73, and 74, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 75, respectively.

[0035] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 80, 81, and 82, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 83, respectively.

[0036] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 58, and 88, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 89, respectively.

[0037] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 41, 94, and 95, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 96, respectively.

[0038] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 101, 102, and 103, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 104, respectively.

[0039] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 109, 110, and 111, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 112, respectively.

[0040] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 117, 118, and 119, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 120, respectively.

[0041] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 125, and 126, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 127, respectively.

[0042] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 41, 132, and 133, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 134, respectively.

[0043] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 57, 139, and 140, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 141, respectively.

[0044] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 146, 147, and 148, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 149, respectively.

[0045] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 102, and 155, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 156, respectively.

[0046] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 101, 161, and 162, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 163, respectively.

[0047] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 168, 169, and 170, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 171, respectively.

[0048] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 81, and 176, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 177, respectively.

[0049] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 182, and 183, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 184, respectively.

[0050] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 189, 190, and 191, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 192, respectively.

[0051] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 197, 198, and 199, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 200, respectively.

[0052] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 205, and 206, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 207, respectively.

[0053] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 212, and 213, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 214, respectively.

[0054] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 189, 219, and 220, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 221, respectively.

[0055] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 227, and 228, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 229, respectively.

[0056] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 57, 132, and 234, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 235, respectively.

[0057] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 41, 240, and 241, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0058] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 109, 50, and 247, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 248, respectively.

[0059] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 253, and 254, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 255, respectively.

[0060] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 260, and 261, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 262, respectively.

[0061] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 132, and 267, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 268, respectively.

[0062] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 274, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0063] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 117, 240, and 280, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0064] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 285, 42, and 286, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 287, respectively.

[0065] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 292, 118, and 293, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0066] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 117, 298, and 299, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0067] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 19, 240, and 304, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0068] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 309, and 310, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 311, respectively.

[0069] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 147, and 316, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 317, respectively.

[0070] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 240, and 322, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0071] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 26, 327, and 328, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 329, respectively.

[0072] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 334, 335, and 336, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 337, respectively.

[0073] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 342, 343, and 344, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 345, respectively.

[0074] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 253, and 350, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 351, respectively.

[0075] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 356, 182, and 357, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 358, respectively.

[0076] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 285, 363, and 364, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 365, respectively.

[0077] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 370, 50, and 371, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 372, respectively.

[0078] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 117, 240, and 377, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0079] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 154, 386, and 386, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 384, respectively.

[0080] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 117, 240, and 389, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 242, respectively.

[0081] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 394, and 395, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 396, respectively.

[0082] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 401, 50, and 402, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 403, respectively.

[0083] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 58, and 408, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 409, respectively.

[0084] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 454, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0085] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 457, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0086] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 460, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0087] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 463, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0088] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 466, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0089] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 469, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0090] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 472, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0091] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 475, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0092] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 478, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0093] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 481, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0094] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 484, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0095] An embodiment of the disclosure relates to an antibody, antigen-binding fragment, or polypeptide comprising a heavy chain variable region having HCDR1, HCDR2, and HCDR3, and a light chain variable region having LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 226, 273, and 487, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 275, respectively.

[0096] The present disclosure relates to SEQ ID NOs:7 and 8, SEQ ID NOs:15 and 16, SEQ ID NOs:22 and 23, SEQ ID NOs:30 and 31, SEQ ID NOs:37 and 38, SEQ ID NOs:45 and 46, SEQ ID NOs:53 and 54, SEQ ID NOs:61 and 62, SEQ ID NOs:68 and 69, SEQ ID NOs:76 and 77, SEQ ID NOs:84 and 85, SEQ ID NOs:90 and 91, SEQ ID NOs:97 and 98, SEQ ID NOs:105 and 106, SEQ ID NOs:113 and 114, SEQ ID NOs:121 and 122, SEQ ID NOs:128 and 129, SEQ ID NOs:135 and 136, SEQ ID NOs:142 and 143, SEQ ID NOs:150 and 151. , SEQ ID NO:157 and 158, SEQ ID NO:164 and 165, SEQ ID NO:172 and 173, SEQ ID NO:178 and 179, SEQ ID NO:185 and 186, SEQ ID NO:193 and 194, SEQ ID NO:201 and 202, SEQ ID NO:208 and 209, SEQ ID NO:215 and 216, SEQ ID NO:222 and 223, SEQ ID NO:230 and 231, SEQ ID NO:236 and 237, SEQ ID NO:243 and 244, SEQ ID NO:249 and 250, SEQ ID NO:256 and 257, SEQ ID NO:263 and 264, SEQ ID NO:269 and 270, SEQ ID NO:276 and 277, Sequence numbers 281 and 282, SEQ ID NOs: 288 and 289, SEQ ID NOs: 294 and 295, SEQ ID NOs: 300 and 301, SEQ ID NOs: 305 and 306, SEQ ID NOs: 312 and 313, SEQ ID NOs: 318 and 319, SEQ ID NOs: 323 and 324, SEQ ID NOs: 330 and 331, SEQ ID NOs: 338 and 339, SEQ ID NOs: 346 and 347, SEQ ID NOs: 352 and 353, SEQ ID NOs: 359 and 360, SEQ ID NOs: 366 and 367, SEQ ID NOs: 373 and 374, SEQ ID NOs: 378 and 379, SEQ ID NOs: 385 and 386, SEQ ID NOs: 390 and 391, SEQ ID NOs: Nos. 397 and 398, 404 and 405, 410 and 411, 455 and 277, 458 and 277, 461 and 277, 464 and 277, 467 and 277, 470 and 277, 473 and 277, 476 and 277, 479 and 277, 482 and 277, 485 and 277, or 488 and 277.

[0097] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for any measuring or quantitating method.

[0098] The words "a" or "an," when used in conjunction with the term "comprising," may mean "one," but are also consistent with the meanings of "one or more," "at least one," and "one or more."

[0099] The term "and / or" means "and" or "or." To illustrate, A, B, and / or C includes A only, B only, C only, A in combination with B, A in combination with C, B in combination with C, or A in combination with B and C. In other words, "and / or" functions as an inclusive or.

[0100] The words "comprising" (and any form of "including", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "including", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0101] The compositions and methods of use can "comprise", "consist essentially of", or "consist of" any of the components or steps disclosed throughout this specification. Compositions and methods that "consist essentially of" any of the disclosed components or steps limit the claims to certain materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. As used in this specification and claims, the words "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "comprising" such as "includes" and "include"), or "containing" (and any form of "comprising" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited components or method steps. It is intended that embodiments or aspects described herein in the context of the term "comprising" may also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0102] "Individual," "subject," and "patient" are used interchangeably and can refer to a human or non-human.

[0103] It is specifically contemplated that any limitations discussed with respect to one embodiment or aspect of the invention may also be applied to other embodiments or aspects of the invention. Moreover, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to make or utilize any composition of the invention. An embodiment or aspect described with respect to one aspect of the disclosure may be applied to other aspects of the disclosure as well, and vice versa. For example, any step in a method described herein may be applied to any other method. Furthermore, any method described herein may have the exclusion of any step or combination of steps. Aspects of the embodiments shown in the examples are also embodiments that can be implemented in the context of embodiments discussed elsewhere in different examples or discussed elsewhere in this application (e.g., Summary of the Invention, Detailed Description, Claims, and Brief Description of the Figures).

[0104] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as the "use" of any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0105] Other objects, features and advantages of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

[0106] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0107] [Figure 1]Figure 1. In vitro validation of specific binding of GHR Fab by protein ELISA. ELISA detection of FAB incubated with recombinant GHR-Fc (biotinylated) in streptavidin-coated plates and detected by a secondary anti-human Fab antibody conjugated to HRP. [Diagram 2] Figure 2. Epitope determination by SPR. Figure 2 shows that epitope binning of GHR FABs by SPR identified two major epitopes. Sensorgrams of representative FABs for each epitope are shown. [Figure 3A] Figure 3A. Validation of Fab binding to human cells expressing GHR. Figure 3A shows quantification of binding by MFI on MDA-MB-435 cells. GHR FAB was added at 200 nM and detected by flow cytometry using an anti-human Fab antibody conjugated to Alexa Fluor® 647. Figure 3B shows quantification of binding by MFI on IM9 cells. GHR FAB was added at 200 nM and detected by flow cytometry using an anti-human Fab antibody conjugated to Alexa Fluor® 647. Data are presented as mean ± SD of three independent experiments (n=3). [Figure 3B] Figure 3B. Validation of Fab binding to human cells expressing GHR. Figure 3A shows quantification of binding by MFI on MDA-MB-435 cells. GHR FAB was added at 200 nM and detected by flow cytometry using an anti-human Fab antibody conjugated to Alexa Fluor® 647. Figure 3B shows quantification of binding by MFI on IM9 cells. GHR FAB was added at 200 nM and detected by flow cytometry using an anti-human Fab antibody conjugated to Alexa Fluor® 647. Data are presented as mean ± SD of three independent experiments (n=3). [Figure 4]Figure 4. Single-point phage ELISA of binders selected by additional phage display campaigns. Clones with strong signals for GHR were selected for further sequencing and characterization. Data points represent the mean from three independent experiments. Error bars represent SD from the mean. [Figure 5A] Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 5B] Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 5C]Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 5D] Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 5E] Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 5F]Figure 5A-F. Characterization of GHR_TS23 binding to GHR from different species. (A) SPR sensorgram shows interaction with human GHR. (B) SPR sensorgram shows interaction with cynomolgus monkey GHR. (C) SPR sensorgram shows interaction with rabbit GHR. (D) SPR sensorgram shows interaction with porcine GHR. (E) SPR sensorgram shows interaction with mouse GHR. (F) SPR sensorgram shows interaction with rat GHR. Fab was serially diluted 2-fold starting at 50 nM. [Figure 6A] Figure 6A-D. GHR Fab as a GH antagonist. (A) Model of a competitive multipoint ELISA experiment. An affinity-improved form of GH (B2036) was incubated with GHR pre-immobilized on a plate. A multipoint ELISA with GHR Fab was performed to verify the ability of the antibody to displace GH from the binding site of the receptor. (B) Competition results show that the selected GHR Fab is highly efficient at displacing GH in the receptor complex. (C) Figures 6C and 6D represent the reverse experiment, where a labeled GHR Fab is pre-bound to the receptor complex and challenged by adding either GH or B2036. Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of GH. (D) Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of B2036. [Figure 6B]Figure 6A-D. GHR Fab as a GH antagonist. (A) Model of a competitive multipoint ELISA experiment. An affinity-improved form of GH (B2036) was incubated with GHR pre-immobilized on a plate. A multipoint ELISA with GHR Fab was performed to verify the ability of the antibody to displace GH from the binding site of the receptor. (B) Competition results show that the selected GHR Fab is highly efficient at displacing GH in the receptor complex. (C) Figures 6C and 6D represent the reverse experiment, where a labeled GHR Fab is pre-bound to the receptor complex and challenged by adding either GH or B2036. Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of GH. (D) Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of B2036. [Figure 6C] Figure 6A-D. GHR Fab as a GH antagonist. (A) Model of a competitive multipoint ELISA experiment. An affinity-improved form of GH (B2036) was incubated with GHR pre-immobilized on a plate. A multipoint ELISA with GHR Fab was performed to verify the ability of the antibody to displace GH from the binding site of the receptor. (B) Competition results show that the selected GHR Fab is highly efficient at displacing GH in the receptor complex. (C) Figures 6C and 6D represent the reverse experiment, where a labeled GHR Fab is pre-bound to the receptor complex and challenged by adding either GH or B2036. Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of GH. (D) Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of B2036. [Figure 6D]Figure 6A-D. GHR Fab as a GH antagonist. (A) Model of a competitive multipoint ELISA experiment. An affinity-improved form of GH (B2036) was incubated with GHR pre-immobilized on a plate. A multipoint ELISA with GHR Fab was performed to verify the ability of the antibody to displace GH from the binding site of the receptor. (B) Competition results show that the selected GHR Fab is highly efficient at displacing GH in the receptor complex. (C) Figures 6C and 6D represent the reverse experiment, where a labeled GHR Fab is pre-bound to the receptor complex and challenged by adding either GH or B2036. Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of GH. (D) Competition results show no displacement of GHR Fab from the receptor by increasing concentrations of B2036. [Figure 7] Figure 7. Fab inhibitory activity. pSTAT5 flow cytometry shows efficient GH-mediated signaling inhibition upon treatment with GHR Fab in vitro. Background pSTAT5 can be observed in samples indicated as "cells". Data points represent the mean from three independent experiments. Error bars represent the SD from the mean. [Figure 8] Figure 8. GHR_TS23 Fab inhibitory activity. The improved inhibitory activity of lead candidate GHR_TS23 upon reconstitution into IgG was assessed using a multipoint assay. As a result, no signaling was observed when cells were treated with GHR_TS23 IgG at a low concentration of 3 nM. [Figure 9A]Figure 9A-B. GHR_TS23 CDR-H3 soft randomization. (A) Model of the soft randomization strategy. The wild-type CDR-H3 sequence of GHR_TS23 used for preparation of the phage library is shown in red. The phage library was designed to display 50% wild-type sequence and 50% all other amino acids at each position in the CDR-H3. Phage display was then performed using human / pig / rabbit GHR as targets to determine the essential amino acids located at the CDR-H3 of GHR_TS23 Fab. (B) WebLogo plot shows sequence variants generated from phage display using soft randomized GHR_TS23. [Figure 9B] Figure 9A-B. GHR_TS23 CDR-H3 soft randomization. (A) Model of the soft randomization strategy. The wild-type CDR-H3 sequence of GHR_TS23 used for preparation of the phage library is shown in red. The phage library was designed to display 50% wild-type sequence and 50% all other amino acids at each position in the CDR-H3. Phage display was then performed using human / pig / rabbit GHR as targets to determine the essential amino acids located at the CDR-H3 of GHR_TS23 Fab. (B) WebLogo plot shows sequence variants generated from phage display using soft randomized GHR_TS23. [Figure 10] Figure 10. Developability of GHR_TS23. The SEC profile shows that the developability of the generated GHR_TS23.1 Fab is improved over wild-type GHR_TS23. [Figure 11] Figure 11. Single-point phage ELISA of soft randomized GHR_TS23 Fab mutants. Wild-type GHR_TS23 results are shown in red. The majority of selected mutants have improved binding to GHR. [Figure 12A]Figure 12A-B. Biophysical characterization of GHR_TS23.1 Fab. (A) SPR sensorgrams show the interaction of GHR_TS23.1 with human GHR. (B) SPR single-injection sensorgrams show that binding to human GHR is lost when essential non-randomized positions in CDR-H3 are mutated. [Figure 12B] Figure 12A-B. Biophysical characterization of GHR_TS23.1 Fab. (A) SPR sensorgrams show the interaction of GHR_TS23.1 with human GHR. (B) SPR single-injection sensorgrams show that binding to human GHR is lost when essential non-randomized positions in CDR-H3 are mutated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] Growth hormone receptor (GHR) belongs to the type I cytokine receptor family and is a master regulator of developmental, cellular, and metabolic processes. Dysregulation of GHR signaling is implicated in many human diseases, including acromegaly, cancer, aging, and longevity. Thus, inhibition of GHR signaling may provide therapeutic benefit in many pathological conditions. As disclosed herein, antibody phage display was used to generate antibody fragments (FABs) and antibodies (IgGs) that inhibit GHR receptor signaling by binding to the extracellular domain, competing with GH binding, and inhibiting receptor activation. Thus, disclosed are compositions comprising various targeting molecules (e.g., antibodies, antibody fragments, antibody-like molecules, antibody drug conjugates, chimeric antigen receptors, BiTES, etc.) that can target GHR, as well as methods of using such molecules for the detection, diagnosis, and treatment of conditions associated with GHR, such as acromegaly and cancer.

[0109] I. Antibodies Aspects of the present disclosure relate to antibodies, antigen-binding fragments thereof, or polypeptides capable of specifically binding to GHR.

[0110] The term "antibody" refers to an intact immunoglobulin of any isotype or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, including chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins and polypeptides containing antibody CDR domains that retain antigen-binding activity.

[0111] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent such as an antibody. An antigen can have one or more epitopes capable of interacting with different antibodies.

[0112] The term "epitope" includes any region or portion of a molecule capable of eliciting an immune response by binding to an immunoglobulin or T-cell receptor. Epitopic determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture.

[0113] Epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art (Johan Rockberg and Johan Nilvebrant, Ed., 2018), including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays (see, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986)). In addition, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0114] The term "immunogenic sequence" refers to a molecule that contains the amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term "immunogenic composition" refers to a composition that contains at least one immunogenic molecule (such as an antigen or carbohydrate).

[0115] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies naturally occurring in camelids, which may contain only heavy chains. The antibodies disclosed herein may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or mouse source, and the constant regions may be derived from a different animal source, such as human. Antibodies or binding fragments may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term "antibody" includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013;4:302;2013).

[0116] The term "light chain" includes full-length light chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of about 25,000 daltons and include a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classes of light chains, identified as kappa (K) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. A VL fragment may further include a light chain constant region sequence. The light chain variable region domain is at the amino-terminus of the polypeptide.

[0117] The term "heavy chain" includes full-length heavy chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of about 50,000 daltons and contains a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the heavy chain variable region, including the CDRs. A VH fragment may further contain heavy chain constant region sequence. The number of heavy chain constant region domains depends on the isotype. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody is IgM, IgD, IgG, IgA, or IgE, and is defined by the heavy chain present, which can be classified as mu (p), delta (s), gamma (y), alpha (a), or epsilon (a) chains, respectively. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0118] A. Types of Antibodies An antibody may be a whole immunoglobulin of any isotype or classification, a chimeric antibody, or a hybrid antibody having specificity for more than one antigen. Antibodies may also be fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic, or recombinant proteins that act like antibodies by binding to a specific antigen to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.

[0119] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units bound to each other via the constant domains (Fc region or fragment crystallizable region) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein. The term "multimer" refers to an antibody that contains three or more Ig units bound to each other via the constant domains (Fc region) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein.

[0120] The term "bivalent antibody" refers to an antibody that comprises two antigen-binding sites. The two binding sites may have the same antigen specificity or may be bispecific, meaning that the two antigen-binding sites have different antigen specificities.

[0121] Bispecific antibodies are a class of antibodies with two paratopes that have different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies are biparatopic, in which case the bispecific antibodies may specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single domain antibodies, called "nanobodies." Single domain antibodies are sourced and modified from cartilaginous fish and camelids. Nanobodies can be linked by linkers using techniques typical to those of skill in the art, and methods for the selection and linking of nanobodies are described in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which is specifically incorporated herein by reference in its entirety.

[0122] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabodies, or scFv. Diabodies and scFv can be constructed without an Fc region using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is specifically incorporated by reference in its entirety.

[0123] In certain embodiments, the antigen-binding domain may be multispecific or heterospecific by multimerizing with pairs of VH and VL regions that bind different antigens. For example, the antibody may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, embodiments include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof against epitopes and other targets, such as Fc receptors on effector cells.

[0124] In some embodiments, multispecific antibodies can be used and directly linked via short flexible polypeptide chains using routine methods known in the art. One example is a bivalent, bispecific antibody, a diabody, in which the VH and VE domains are expressed on a single polypeptide chain and utilize a linker that is too short to allow pairing between the domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen binding sites. Linker functions are applicable to triabodies, tetrabodies, and higher order antibody multimer embodiments (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0125] In contrast to bispecific whole antibodies, bispecific diabodies can be advantageous because they can be easily constructed and expressed in E. coli. Diabodies (and other polypeptides such as antibody fragments) with appropriate binding specificity can be easily selected from libraries using phage display (WO94 / 13804). Libraries can be created where one arm of the diabody is kept constant, e.g., with specificity for a protein, and the other arm is varied to select for antibodies with appropriate specificity. Bispecific whole antibodies can also be produced by alternative engineering methods described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in its entirety.

[0126] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities, see, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0127] The portion of the Fv fragment of an antibody molecule that binds with high specificity to an epitope of an antigen is referred to herein as the "paratope". The paratope is composed of amino acid residues that contact the epitope of the antigen and promote antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains, the VH and VL domains, in a dimerization configuration. The primary structure of each variable domain comprises three hypervariable loops separated by and flanked by framework regions (FR). The hypervariable loops are the regions of greatest primary sequence variability in antibody molecules from any mammal. The term hypervariable loop may be used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have a high degree of primary sequence similarity / consensus. The consensus of the framework regions can be used by the skilled artisan to identify both the framework regions and the hypervariable loops (or CDRs) interspersed between the framework regions. The hypervariable loops are given identifiers that distinguish their location in the polypeptide and in which domain they are found. The CDRs of the VL domain are identified as LI, L2, and L3, with LI being the most distal and L3 being closest to the CL domain. The CDRs are sometimes named CDR-L1 (or LCDR1), CDR-L2 (or LCDR2), and CDR-L3 (or LCDR3). L3 (CDR-L3) is generally the most variable region of all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and are separated from each other by framework regions. The amino terminus (N-terminus) of the VL chain is designated FR1. The region identified as FR2 is between the LI and L2 hypervariable loops. FR3 is between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which comprises three CDRs identified as CDR-H1 (or HCDR1), CDR-H2 (or HCDR2) and CDR-H3 (or HCDR3).The majority of amino acid residues in the variable domains or Fv fragments (VH and VL) are part of framework regions (about 85%). The three-dimensional or tertiary structure of an antibody molecule is such that the framework regions are more internal to the molecule and provide most of the structure, while the CDRs are on the outer surface of the molecule.

[0128] Several methods have been developed and can be used by one of skill in the art to precisely identify the amino acids that make up each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions, and thus identify the CDRs that are located between the framework regions, although they may vary in length. Three commonly used methods have been developed for the identification of antibody CDRs: Kabat (described in TTWu and EAKabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (described in M.-P.Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative (Described in Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods involves a unique numbering system for identifying the amino acid residues that make up the variable region. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen are present in the CDRs, but in some cases, residues in the framework regions contribute to antigen binding.

[0129] One of skill in the art can use any of several methods to determine the paratope of an antibody. These methods include:

[0130] 1) Computational prediction of the tertiary structure of antibody / epitope binding interactions based on the chemical nature of the amino acid sequences of the antibody variable regions and the composition of the epitope.

[0131] 2) Hydrogen-deuterium exchange and mass spectrometry

[0132] 3) Polypeptide fragmentation and peptide mapping approach: multiple overlapping peptide fragments are generated from a full-length polypeptide and the binding affinity of these peptides to the epitope is assessed.

[0133] 4) Antibody phage display library analysis: antibody Fab fragments encoding mammalian genes are expressed by bacteriophage in such a way that they are incorporated into the phage coat. This population of Fab-expressing phages is then allowed to interact with antigens, which may be immobilized or expressed by different exogenous expression systems. Unbound Fab fragments are washed away, leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be easily isolated and the genes encoding them determined. This approach can also be used for Fv fragments or smaller regions of Fab fragments containing specific VH and VL domains, if desired.

[0134] In certain embodiments, the affinity matured antibody is enhanced by one or more modifications in one or more of its CDRs that result in improved affinity of the antibody for the target antigen compared to the parent antibody without the modification. Certain affinity matured antibodies have nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies are generated by procedures known in the art, for example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, and random mutagenesis of CDR and / or framework residues used in phage display is described by Rajpal et al., PNAS.24:8466-8471 (2005) and Thie et al., Methods Mol Biol.525:309-22 (2009) in combination with computational methods shown in Tiller et al., Front.Immunol.8:986 (2017).

[0135] Chimeric immunoglobulins are the product of fusion genes derived from different species. "Humanized" chimeras generally have framework regions (FR) derived from a human immunoglobulin and one or more CDRs derived from a non-human source.

[0136] In certain embodiments, a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence from another particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods relating to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), each of which is specifically incorporated herein by reference in its entirety. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated by reference herein for all purposes.

[0137] In some embodiments, the function of the chimeric antibody is optimized and immunogenicity is reduced by minimizing antibody polypeptide sequences derived from non-human species. Certain amino acid residues from non-antigen recognition regions of the non-human antibody are modified to be homologous to corresponding residues in human antibodies or isotypes. One example is a "CDR-grafted" antibody, in which the antibody comprises one or more CDRs derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the antibody chain is identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass. For use in humans, the V-regions, consisting of CDR1, CDR2, and partial CDR3 for both the light and heavy chain variable regions from a non-human immunoglobulin, are grafted onto human antibody framework regions, replacing the naturally occurring antigen receptor of the human antibody with the non-human CDRs. In some cases, the corresponding non-human residues are replaced with framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may contain residues not found in the recipient antibody or donor antibody to further improve performance. A humanized antibody can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23:1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0138] Intrabodies are immunoglobulins that are located intracellularly and bind to intracellular antigens, as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0139] Polyclonal antibody preparations typically contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host, such as a rabbit or goat, is generally immunized with an antigen or an antigen fragment together with an adjuvant (optionally bound to a carrier). Antibodies against the antigen are then collected from the host's serum. Polyclonal antibodies can be affinity purified against the antigen to render them monospecific.

[0140] Monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies derived from only one parent cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0141] B. Functional Antibody Fragments and Antigen-Binding Fragments 1. Antigen-binding fragment Particular aspects relate to antibody fragments, such as antibody fragments that bind to GHR. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to antigen. These fragments are composed of various arrangements of variable region heavy chains (VH) and / or light chains (VL), and in some embodiments, constant region heavy chain 1 (CHI) and light chains (CL). In some embodiments, they lack the Fc region, which is composed of heavy chain 2 (CH2) and heavy chain 3 (CH3) domains. Embodiments of antigen-binding fragments and modifications thereof may include (i) Fab fragment species composed of VL, VH, CL, and CHI domains, (ii) Fd fragment species composed of VH and CHI domains, (iii) Fv fragment species composed of VH and VL domains, (iv) single domain fragment species composed of a single VH or VL domain, dAb (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003), and (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and in Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015), each of which is incorporated by reference.

[0142] Antigen-binding fragments also include fragments of antibodies that retain at least, at most, or exactly one, two, or three complementarity determining regions (CDRs) from a light chain variable region. Fusions of CDR-containing sequences with an Fc region (or its CH2 or CH3 regions) are included within this definition, e.g., scFvs fused directly or indirectly to an Fc region are included herein.

[0143] The term Fab fragment ("Fab" or "FAB") refers to a monovalent antigen-binding fragment of an antibody that contains the VL, VH, CL, and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment contains the VL, VH, CL, and CH1 domains and all or a portion of the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody that contains two Fab' fragments linked by a disulfide bond at the hinge region. An F(ab')2 fragment, for example, contains all or a portion of two VH and VL domains and can further contain two CL domains and all or a portion of a CHI domain.

[0144] The term Fd fragment refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the VH, including the CDRs. The Fd fragment may further contain a CH1 region sequence.

[0145] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of the VL and VH, and that is devoid of the CL and CH1 domains. The VL and VH include, for example, the CDRs. A single-chain antibody (sFv or scFv) is an Fv molecule in which the VL and VH domains are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain that includes an oligomerization domain at the C-terminus, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain includes a self-associating alpha-helix, such as a leucine zipper, and can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also called "miniantibodies" or "minibodies."

[0146] A single domain antibody is an antigen-binding fragment that contains only a VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same antigen or different antigens.

[0147] 2. Fragment antigen-binding region, Fab The Fab polypeptides of the present disclosure include Fab antigen-binding fragments of antibodies. Unless otherwise specified, the term "Fab" refers to a polypeptide excluding the Fc portion of an antibody. The Fab may be joined to a polypeptide that includes other components such as additional antigen-binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and accessory proteins. Fab polypeptides can be generated using conventional techniques known in the art and are well described in the literature.

[0148] 3.Crystallizable fragment region Fc The Fc region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. As used herein, the term "Fc polypeptide" includes native and mutated forms of polypeptides derived from the Fc region of an antibody. It includes truncated forms of such polypeptides, including the hinge region that promotes dimerization.

[0149] C. Polypeptides with Antibody CDRs and Scaffolding Domains Presenting the CDRs An antigen-binding peptide scaffold, such as a complementarity determining region (CDR), is used to generate a protein-binding molecule according to an embodiment. In general, a person skilled in the art can determine the type of protein scaffold to graft at least one of the CDRs. It is known that the scaffold must meet a number of criteria, such as optionally having good phylogenetic conservation, a known three-dimensional structure, a small size, little or no post-translational modification, and / or being easy to manufacture, express, and purify (Skerra, J Mol Recognit, 13:167-87 (2000)).

[0150] Protein scaffolds can be sourced from, but are not limited to, fibronectin type III FN3 domain (known as "monobody"), fibronectin type III domain 10, lipocalins, anticalins, the Z domain of Staphylococcus aureus protein A, thioredoxin A, or proteins with repeat motifs such as "ankyrin repeats", "armadillo repeats", "leucine-rich repeats", and "tetratricopeptide repeats". Such proteins are described in U.S. Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. WO2006 / 056464, each of which is incorporated herein by reference in its entirety. Scaffolds derived from venoms of scorpions, insects, plants, mollusks, etc. and protein inhibitors of neuronal NO synthase (PIN) may be used.

[0151] D. Antibody binding The term "selective binding agent" refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab', F(ab')2, single chain antibodies, peptides, peptide fragments, and proteins.

[0152] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic bonds, and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). "Immunologically reactive" means that a selective binding agent or an antibody of interest binds to an antigen present in a biological sample. The term "immunocomplex" refers to the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.

[0153] 1. Avidity The term "affinity" refers to the strength with which an antibody or selective binding agent binds to an epitope. In antibody binding reactions, this is expressed as an affinity constant (Ka or ka, sometimes referred to as binding constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of an antibody to an antigen to that of an unrelated amino acid sequence. Affinity can be expressed, for example, as a 20-fold greater binding ability of an antibody to an antigen versus an unrelated amino acid sequence. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or selective binding agents.

[0154] There are several experimental methods that can be used by those skilled in the art to evaluate the binding affinity of any given antibody or selective binding agent to an antigen. This is generally done by measuring the equilibrium dissociation constant (KD or Kd) using the formula KD=koff / kon=[A][B] / [AB]. The term koff is the dissociation rate between the antibody and the antigen per unit time and is related to the concentration of the antibody and antigen present in solution in unbound form at equilibrium. The term kon is the binding rate between the antibody and the antigen per unit time and is related to the concentration of the bound antigen-antibody complex at equilibrium. The unit used to measure KD is mol / L (molar concentration or M), i.e., concentration. The Ka of an antibody is the inverse of the KD and is determined by the formula Ka=1 / KD. Examples of some experimental methods that can be used to determine KD values ​​are enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE). The affinity constant (Ka) of an antibody is the inverse of the KD and is determined by the formula Ka=1 / KD.

[0155] Antibodies that are considered useful in certain embodiments have a molecular weight of at least or at most about 10 6 , 10 7 , 10 8 , 10 9, or 10 10 In some embodiments, the antibody may have an affinity constant (Ka) of at least or at most about 10 -6 , 10 -7 , 10 -8 , 10 -9 , or 10 -10 The antibodies of the invention may have a dissociation constant of 0.1 to 10 M, 0.2 to 10 M, or any range derivable therein. These values ​​are reported for the antibodies discussed herein, and the same assays may be used to assess the binding properties of such antibodies. The antibodies of the invention have a dissociation constant (KD) ≦10 -8 An antibody is said to "specifically bind" to its target antigen if its KD ≤ 5 × l0 -9 "High affinity" in the case of M and KD ≤ 5 x 10 -10 In the case of M, it binds specifically to the antigen with "very high affinity."

[0156] 2. Epitope specificity An epitope of an antigen is a particular region of an antigen to which an antibody has binding affinity. In the case of a protein or polypeptide antigen, an epitope is a particular residue (or designated amino acid or protein segment) to which an antibody binds with high affinity. An antibody does not necessarily contact every residue in a protein. Nor does substitution or deletion of every amino acid in a protein necessarily affect binding affinity. For purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen to which B cells and / or T cells respond or recognize. Polypeptide epitopes are formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a polypeptide. An epitope typically contains at least 3 and typically 5-10 amino acids in a unique spatial arrangement.

[0157] The epitope specificity of an antibody can be determined by a variety of methods. For example, one approach involves testing a collection of overlapping peptides of 15 amino acids that span the entire sequence of the protein and differ in a small number of amino acid increments (e.g., 3-30 amino acids). The peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one end of the peptide. This process can affect the affinity of the antibody for the epitope, and therefore different samples of the same peptide can be biotinylated at the N-terminus and C-terminus and immobilized in separate wells for comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides that terminate at specific amino acids of interest can be included. This approach is useful for identifying end-specific antibodies to internal fragments. The antibody or antigen-binding fragment is screened for binding to each of the various peptides. The epitope is defined as a segment of amino acids common to all peptides to which the antibody exhibits high affinity binding.

[0158] 3. Modification of antibody antigen-binding domain It is understood that the antibodies of the present invention may be modified such that the antibodies are substantially identical to an antibody polypeptide sequence or fragment thereof and still bind to an epitope of the present invention. Polypeptide sequences are "substantially identical" when optimally aligned using programs such as Clustal Omega, IGBLAST, GAP, or BESTFIT using default gap weights and have at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, or any range therein.

[0159] As discussed herein, minor variations in the amino acid sequence of an antibody or antigen-binding region thereof are contemplated to be encompassed by the present invention so long as the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% sequence identity. In particular, conservative amino acid substitutions are contemplated.

[0160] Conservative substitutions are those made within a family of amino acids whose side chains are related. Genetically encoded amino acids are generally classified into families based on the chemical properties of their side chains, e.g., acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). For example, it is reasonable to expect that the isolated replacement of a leucine moiety with an isoleucine or valine moiety, or the similar replacement of an amino acid with a structurally related amino acid of the same family, will not significantly affect the binding and properties of the resulting molecule, especially if the replacement does not involve an amino acid in a framework region. Whether an amino acid change results in a functional peptide can be easily determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, surface plasmon resonance (SPR), or other antibody binding assays can be performed by one of skill in the art to make a quantitative comparison of antigen binding affinity between an unmodified antibody and any polypeptide derivative with conservative substitutions generated by any of a number of methods available to one of skill in the art.

[0161] Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Standard methods for identifying protein sequences that fold into known three-dimensional structures are available to those skilled in the art (Dill and McCallum., Science 338:1042-1046 (2012)). Several algorithms have been developed to predict protein structures and the gene sequences that code for them, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / guide / proteins / ) and the Bioinformatics Resource Portal (on the World Wide Web at expasy.org / proteomics). Thus, the foregoing examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the present invention.

[0162] For example, framework modifications can be made to the antibody to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0163] It is also contemplated that antigen-binding domains may be made multispecific or multivalent by multimerizing them with pairs of VH and VL domains that bind either the same antigen (multivalent) or different antigens (multispecific).

[0164] E. Chemical Modification of Antibodies In some aspects, glycosylation variants of antibodies are also contemplated in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. Glycosylation of a polypeptide can be altered, for example, by modifying one or more glycosylation sites within the polypeptide sequence to increase the affinity of the polypeptide for an antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861). In certain embodiments, the antibody protein variants contain a greater or lesser number of N-linked glycosylation sites than the native antibody. N-linked glycosylation sites are characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that remove or alter this sequence prevent the addition of an N-linked carbohydrate chain present in the native polypeptide. For example, glycosylation can be reduced by deleting Asn or substituting Asn with another amino acid. In other embodiments, one or more new N-linked glycosylation sites are created. Antibodies typically have N-linked glycosylation sites in the Fc region.

[0165] Additional antibody variants include cysteine ​​variants in which one or more cysteine ​​residues in the parent or native amino acid sequence have been deleted or replaced with another amino acid (e.g., serine). Cysteine ​​variants are particularly useful when the antibody needs to be refolded into a biologically active structure. Cysteine ​​variants have fewer cysteine ​​residues than the native antibody, typically an even number to minimize interactions resulting from unpaired cysteines.

[0166] In some embodiments, a polypeptide can be pegylated to extend biological half-life by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG under conditions in which one or more PEG groups are attached to the polypeptide. The pegylation of the polypeptide may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylation of proteins are known in the art and can be applied to the polypeptides of the invention to obtain pegylated derivatives of antibodies. See, for example, EP0154316 and EP0401384. In some embodiments, the antibody is conjugated or linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with a chemical selected from the group consisting of, for example, dextran, poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol. As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins.

[0167] 1. Join Derivatives of the antibodies and antigen-binding fragments described herein are also provided. Derivatized antibodies or fragments thereof may include any molecule or substance that confers desired properties to the antibody or fragment. Derivatized antibodies may include, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharma- ceutical active moiety), or a molecule that enhances the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human, or other in vivo or in vitro use).

[0168] Optionally, the antibody or immunological portion of the antibody can be chemically conjugated to or expressed as a fusion protein with other proteins. In some embodiments, the polypeptide can be chemically modified by conjugating or fusing to a serum protein, such as human serum albumin, to extend the half-life of the resulting molecule. See, for example, EP0322094 and EP0486525. In some embodiments, the polypeptide can be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the onset or progression of a disease and to determine the effectiveness of a given treatment regimen. In some embodiments, the polypeptide can also be conjugated to a therapeutic agent to provide a treatment in combination with the therapeutic effect of the polypeptide. Additional suitable conjugated molecules include ribonucleases (RNases), DNaseI, antisense nucleic acids, inhibitory RNA molecules such as siRNA molecules, immunostimulatory nucleic acids, aptamers, ribozymes, triplex forming molecules, and external guide sequences. Functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity of a target molecule, or they may have de novo activity independent of other molecules.

[0169] In some embodiments, antibodies and antibody-like molecules are disclosed that are linked to at least one agent to form an antibody conjugate or payload. To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or conjugate at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules that have a desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules attached to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands.

[0170] A conjugate A particular example of an antibody conjugate is an antibody conjugate in which the antibody is linked to a detectable label. A "detectable label" is a compound and / or element that can be detected by a particular functional and / or chemical property, by which the antibody can be detected and, if desired, further quantified. Examples of detectable labels include, but are not limited to, radioisotopes, fluorescers, semiconductor nanocrystals, chemiluminescent agents, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin, or haptens), and the like. Specific examples of labels include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas Red, luminol, NADPH, and α- or β-galactosidase. Antibody conjugates include those intended primarily for in vitro use, in which the antibody is linked to a secondary binding ligand and / or an enzyme to generate a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxide, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference. Molecules containing azide groups may also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low intensity ultraviolet light (Potter & Haley, 1983).

[0171] In some embodiments, the immunoconjugate comprises an antibody or an antigen-binding fragment thereof conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin or fragment thereof from bacterial, fungal, plant, or animal origin), or a radioisotope (i.e., a radioconjugate). In this manner, the agent of interest can be directly targeted to a cell bearing a cell surface antigen. The antibody and agent may be associated by non-covalent interactions, such as electrostatic forces, or by covalent bonds. Various linkers known in the art can be used to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein. In one embodiment, the antibody may be conjugated to various therapeutic agents to target cell surface antigens. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins, and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens.

[0172] In antibody drug conjugates (ADCs), an antibody (Ab) is linked to one or more drug moieties (D) via a linker (L). ADCs may be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including (1) reacting a nucleophilic group on an antibody with a bivalent linker reagent to form Ab-L by covalent attachment, followed by reaction with a drug moiety D, and (2) reacting a drug moiety with a bivalent linker reagent to form DL by covalent attachment, followed by reaction with a nucleophilic group on an antibody. Antibody drug conjugates are also made by modifying an antibody to introduce an electrophilic moiety that can react with a nucleophilic substituent on the linker reagent or drug. Alternatively, a fusion protein comprising an antibody and a cytotoxic agent may be made, for example, by recombinant techniques or peptide synthesis. The length of DNA may include respective regions encoding the two portions of the conjugate, adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting of tumor or cancer cells, where the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0173] Examples of antibody-drug conjugates known to those skilled in the art are prodrugs useful for local delivery of cytotoxic or cytostatic agents, i.e., agents that kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systemic administration of these unconjugated drugs can result in unacceptable levels of toxicity to normal cells as well as to target tumor cells (Baldwin et al., Lancet 1:603-5 (1986); Thorpe, (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” In: Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp.475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)).

[0174] In certain embodiments, ADCs include covalent or aggregate conjugates of antibodies or antigen-binding fragments thereof with other proteins or polypeptides, such as by expression of recombinant fusion proteins containing heterologous polypeptides fused to the N-terminus or C-terminus of the antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Fusion proteins containing antibodies may include a peptide (e.g., poly-His) added to facilitate purification or identification of the antibody. Antibody polypeptides may also be linked to a FLAG® (Sigma-Aldrich, St. Louis, Mo.) peptide, as described in Hopp et al., Bio / Technology 6:1204 (1988) and U.S. Pat. No. 5,011,912. Oligomers containing one or more antibody polypeptides may be used as antagonists. Oligomers may be in the form of covalently or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibody polypeptides are contemplated for use. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. In certain embodiments, the oligomer comprises multiple antibody polypeptides linked by covalent or non-covalent interactions between peptide moieties fused to the antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides with properties that promote oligomerization. Leucine zippers and certain polypeptides derived from antibodies are peptides that can promote oligomerization of antibody polypeptides attached thereto, as described in more detail below.

[0175] b. Combined methodology Several methods for the attachment or conjugation of an antibody to its conjugate moiety are known in the art. Some conjugation methods involve the use of metal chelate complexes with organic chelating agents, such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3-6-diphenylglycouril-3, attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each of which is incorporated herein by reference). Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodic acid. Conjugates can also be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, it is contemplated to derivatize immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates made according to this methodology have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules has also been disclosed in the literature, where the effector or reporter molecule is attached to a carbohydrate residue in the Fc region (O'Shannessy et al., 1987).

[0176] II. Antibody production Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutic agents are well known in the art, for example, as disclosed in U.S. Patent Nos. 4,011,308, 4,722,890, 4,016,043, 3,876,504, 3,770,380, and 4,372,745 (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference). These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, hybrid or chimeric antibodies, such as humanized antibodies, modified antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, single domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof that bind to the antigen of interest. In certain aspects, polypeptides, peptides, proteins, and immunogenic fragments thereof for use in various embodiments can also be synthesized in solution or on a solid support according to conventional techniques, see, e.g., Stewart and Young (1984), Tarn et al. (1983), Merrifield (1986), and Barany and Merrifield (1979), each of which is incorporated herein by reference.

[0177] Another way to generate antibodies is by molecular display techniques. These include techniques such as phage display and yeast surface display, which have been the main driving force in their development (Winter, 1994; Boder, 2000; Viti, 2000; Bradbury, 2011). They are capable of displaying single-chain Fv (scFv) and Fab fragments on the surface of microorganisms, linking the phenotype of a variant to its cognate DNA and amino acid sequence (McCafferty, 1990; Kang, 1991; Barbas, 1991). Using these techniques, it is possible to generate libraries of antibody fragments containing billions of variants and to identify variants with the desired binding function from the library in an HTP format.

[0178] In the structure of a standard antibody, six surface loops (three each from the heavy and light chain variable domains) constitute the complementarity determining regions (CDRs). Thus, antibodies and their fragments can be considered as "molecular scaffolds" for displaying CDRs. The sequence diversity of the CDRs is the main source of antibody affinity and specificity. In recombinant antibody libraries, the diversity of the CDRs can be obtained from the innate immune system or generated by random mutagenesis, and both types of Fv libraries have been used successfully (Barbas, 1992; Knappik, 2000).

[0179] Yeast surface display is another important technological development in antibody engineering. Although the library sizes that can be achieved with this technology are much smaller than those of phage display, combining yeast surface display with fluorescence-activated cell sorting (FACS) allows for HTP quantitative characterization of antibody affinity, specificity, and expression efficiency (Boder, 2000). The main strengths of phage display include the large library size and the rapid completion of selection and propagation cycles (less than 2 days). Yeast display offers a highly quantitative readout of binding and expression levels using FACS, as well as the ability to precisely define the stringency of selection based on equilibrium and kinetic binding parameters and expression levels. The integration of these two display methods can provide optimal selection of clones that are not only high affinity binders, but also stable and well-expressing.

[0180] The end product of recombinant antibody screening is a gene encoding Fv and Fab fragments with the desired binding properties. Bacterial expression systems have been established for Fv and Fab with good yields [Borrebaeck, 1992]. Thus, phage display clones can be rapidly reformatted into expression cassettes to generate soluble Fv and Fab fragments.

[0181] Library design - At least 15-20 residues of the CDR residues contribute to the binding of the antibody to the target. This full combinatorial randomization of many sites together far exceeds the diversity limits of phage and yeast display libraries. Because the number of sites cannot be significantly reduced, new library strategies have been developed that reduce the genetic code of the 20 natural amino acids to a smaller subset while maintaining high functional diversity (Fellouse, 2007). These libraries using a significantly reduced genetic code allow the generation of high affinity Fab and scFv molecules. Several reduced genetic code libraries have been constructed and tested, but the concept has not been optimized for different target classes, especially the smaller scaffold camelid VHH and FN3 domains (D2). Screening of these new libraries combines phage display with yeast surface display for optimal efficiency. Humanized antibody scaffold - In molecular display approaches, generally only the variable regions of the antigen binding loops (CDRs) of the Fab domain are diversified. The main architecture of the domain is kept constant. This primary architecture can be based on a fully human antibody scaffold, avoiding the need to humanize the scaffold as required by methods involving animal immunization.

[0182] Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC and affinity chromatography. Monoclonal antibodies may be further screened or optimized for properties related to specificity, avidity, half-life, immunogenicity, binding association, binding dissociation, or overall functional properties related to the treatment of infectious diseases. Thus, monoclonal antibodies may have alterations in the amino acid sequences of the CDRs, including insertions, deletions, or substitutions with conserved or non-conserved amino acids.

[0183] The immunogenicity of certain immunogen compositions can be enhanced by using non-specific stimulators of the immune response, known as adjuvants. Adjuvants that may be used according to embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response that contains killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and / or aluminum hydroxide adjuvant. In addition to adjuvants, it may be desirable to co-administer cimetidine (CIM; 1200 mg / day) (Smith / Kline, PA), low-dose cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines such as β-interferon, IL-2, or IL-12, or biological response modifiers (BRMs) such as genes encoding proteins involved in immune helper function, such as B-7. Phage display systems can be used to grow populations of antibody molecules in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Patent Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol. al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).

[0184] III. Antibodies, Antigen-Binding Fragments, and Polypeptides As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous form of a molecule that occurs naturally in an organism. In some embodiments, a wild type form of a protein or polypeptide is used, while in many embodiments of the present disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms may be used interchangeably. A "modified protein" or "modified polypeptide" or "mutant" refers to a protein or polypeptide that has an altered chemical structure, particularly an amino acid sequence, relative to a wild type protein or polypeptide. In some embodiments, a modified / mutated protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / mutated protein or polypeptide may be altered with respect to one activity or function, but retains the wild type activity or function in other respects, such as immunogenicity. The term polypeptide may also include antibody fragments as described herein, as well as antibody domains such as HCDR1 (heavy chain complementarity determining region 1), HCDR2 (heavy chain complementarity determining region 2), HCDR3 (heavy chain complementarity determining region 3), LCDR1 (light chain complementarity determining region 1), LCDR2 (light chain complementarity determining region 2), LCDR3 (light chain complementarity determining region 3), HFRW1 (heavy chain framework region 1), HFRW2 (heavy chain framework region 2), HFRW3 (heavy chain framework region 3), HFRW4 (heavy chain framework region 4), LFRW1 (light chain framework region 1), LFRW2 (light chain framework region 21), LFRW3 (light chain framework region 3), LFRW4 (light chain framework region 4), VH (heavy chain variable region), VL (light chain variable region), CH (heavy chain variable region), CH (heavy chain constant region), or CL (light chain constant region).

[0185] When a protein is specifically mentioned herein, it generally refers to a native (wild type) or recombinant (modified) protein, or a protein with, optionally, a signal sequence removed. A protein may be directly isolated from an organism in which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a particular polypeptide, generally referring to a polypeptide that is produced from or is a product of replication of in vitro manipulated nucleic acid molecules.

[0186] In certain embodiments, the size of the antibody, antigen-binding fragment, protein, or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 107, 108, 109 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, The domain may include, but is not limited to, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any derivable range therein, or derivatives of the corresponding amino acid sequences described or referenced herein. The polypeptides may be mutated by being truncated to be shorter than the corresponding wild type, and may also be modified by fusing or conjugating heterologous protein or polypeptide sequences having a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any discrete functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids having a structure or function that can be recognized by one of skill in the art.

[0187] Antibodies, antigen-binding fragments, polypeptides, proteins of the disclosure, or polynucleotides encoding such polypeptides or proteins, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) or more mutated amino acids or or may contain at least or up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 1 44, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 5, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids or derivatives therein The compound may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to any range derivable therein.

[0188] In some embodiments, the antibody, antigen-binding fragment, protein, or polypeptide is selected from the group consisting of 1-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 11 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 1 72, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 20 3, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 amino acids (or any derivable range therein).

[0189] In some embodiments, the antibody, antigen-binding fragment, or polypeptide is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 1 73, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 20 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) contiguous amino acids.

[0190] In some embodiments, the antibody, antigen-binding fragment, protein, or polypeptide is at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable therein) identical to one of SEQ ID NOs: 1-502. Similar, identical, or homologous to at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 1 40, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 1, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、929、930、931、932、933、934、935、936、937、938、939、940、941、942、943、944、945、946、947、948、949、950、951、952、953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) contiguous amino acids.

[0191] In some embodiments, any one of SEQ ID NOs: 1-502, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 , 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 2 09, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 24 0, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 8 34, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 86 5, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896 , 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 9 59, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 positions and at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 7618、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、31、 8、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、815、816、817、818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 86 8, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 9 19, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969 , 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any range derivable therein) consecutive amino acids or nucleotides.

[0192] In some embodiments, the heavy chain, light chain, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFRW1, HFRW2, HFRW3, HFRW4, LFRW1, LFRW2, LFRW3, or LFRW4 identified in Table 1, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 of SEQ ID NOs: 1-502. , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 1 20, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 1 51, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 199, 200, 2, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 , 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292 , 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 34 0, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 3 The amino acid at position 88, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or 400 is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0193] In some embodiments, a polypeptide (e.g., antibody, antibody fragment, Fab, etc.) of the disclosure is at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 1-502. In some embodiments, a polypeptide comprises one, two, and / or three CDRs from one of SEQ ID NOs: 1-502. The CDRs may be as determined by Kabat, IMGT, or Chothia. In further embodiments, the polypeptide may have CDRs with one, two, and / or three amino acid changes (e.g., one or two amino acid additions, one or two amino acid deletions, substitutions) with respect to one, two, or three of these CDRs. In some aspects, the polypeptide additionally or alternatively comprises variable region sequences that are not CDR sequences, i.e., amino acid sequences that are at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to the amino acid sequences of the variable region framework.

[0194] The CDRs, from amino terminus to carboxy terminus, are CDR1, CDR2, and CDR3. In some embodiments, a polypeptide may have CDRs with 1, 2, and / or 3 amino acid changes (e.g., addition of 1 or 2 amino acids, deletion, substitution of 1 or 2 amino acids) with respect to CDR1, CDR2, or CDR3. In some embodiments, the CDRs of SEQ ID NOs: 1-502 may further comprise 1, 2, 3, 4, 5, or 6 additional amino acids at the amino or carboxy terminus of the CDR. The additional amino acids may be from the heavy and / or light chain framework regions of SEQ ID NOs: 1-502, which are shown as immediately adjacent to the CDR. Thus, embodiments relate to polypeptides comprising HCDR1 (i.e., CDR-H1), HCDR2 (i.e., CDR-H2), HCDR3 (i.e., CDR-H3), LCDR1 (i.e., CDR-L1), LCDR2 (i.e., CDR-L2), and / or LCDR3 (i.e., CDR-L3) having at least or at most or exactly 1, 2, 3, 4, 5, 6, or 7 amino acids at the amino terminus of the CDR or the carboxy terminus of the CDR, where the additional amino acids are 1, 2, 3, 4, 5, 6, or 7 amino acids of Table 1 or SEQ ID NOs: 1-502 shown as immediately adjacent to the CDR. Other embodiments relate to antibodies comprising one or more CDRs, where the CDRs are fragments of Table 1 or SEQ ID NOs: 1-502, and the fragments may lack 1, 2, 3, 4, or 5 amino acids from the amino terminus or carboxy terminus of the CDR. In some embodiments, the CDRs may lack 1, 2, 3, 4, 5, 6, or 7 amino acids from the carboxy terminus and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from framework regions at the amino terminus of the CDR. In some embodiments, the CDRs may lack 1, 2, 3, 4, 5, 6, or 7 amino acids from the amino terminus and may further comprise 1, 2, 3, 4, 5, 6, 7, or 8 amino acids from framework regions at the carboxy terminus of the CDR. In further embodiments, the antibodies may alternatively or additionally be humanized in regions outside the CDRs and / or the variable regions.In some embodiments, the polypeptide additionally or alternatively comprises an amino acid sequence of a variable region that is not a CDR sequence, i.e., an amino acid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical or homologous to an amino acid sequence of a variable region framework.

[0195] In other embodiments, the polypeptide or protein comprises one, two, three, four, five, or six CDRs from either or both of the light and heavy chain variable regions of Table 1 or SEQ ID NOs: 1-502, and the one, two, three, four, five, or six CDRs may have one, two, and / or three amino acid changes with respect to those CDRs. In some embodiments, some or all of the antibody sequence outside of the variable regions is humanized. The protein may comprise one or more polypeptides. In some aspects, the protein may comprise one or two polypeptides similar to a heavy chain polypeptide and / or one or two polypeptides similar to a light chain polypeptide.

[0196] Nucleotide sequences of various genes, as well as protein, polypeptide and peptide sequences, have been previously published and can be found in recognized computerized databases.Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web).The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0197] It is contemplated that compositions of the present disclosure will have from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may be at least about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). 5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), or up to about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0198] IV. Growth Hormone Receptor (GHR) Aspects of the present disclosure relate to compositions and methods for identifying, detecting, and targeting growth hormone receptors. Growth hormone receptors (GHRs, also known as somatotropin receptors) are key regulators of organismal development and growth. 1,2It is a member of the type I cytokine receptor family and is specifically and uniquely activated by growth hormone (GH). Upon secretion, GH binds to GHR at site 1 on one of the subunits and subsequently binds to site 2 on the other subunit using a distinct protein interface, resulting in receptor dimerization and subsequent conformational changes that lead to activation of the associated tyrosine kinase Janus kinase 2 (JAK2) and downstream activation of signal transduction and activator of transcription (STAT) factors. 3、4 STAT5 is phosphorylated on several residues, which serves as a good downstream marker of GHR signaling. 5 Activation of GHR signaling alters cell metabolism, proliferation, survival, migration, and release of cytokines such as IGF1, thereby altering growth and development at the organismal level. Consistent with its biological functions, GHR signaling has been implicated in many human conditions, including developmental delay, cancer, aging, longevity, and disorders such as acromegaly. 6~9 .

[0199] GHR is encoded by the gene GHR. An example of the mRNA sequence encoding human GHR is available as NCBI RefSeq NM_000163. An example of the protein sequence of human GHR is available as NCBI RefSeq NP_000154.

[0200] Specific examples of polypeptide, antibody, and antigen-binding fragment sequences are provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

Table 1-67

Table 1-68

Table 1-69

Table 1-70

Table 1-71

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

[0201] 1. Mutant Polypeptides The following discusses changing the amino acid subunits of proteins to create equivalent or improved second generation mutant polypeptides or peptides. For example, certain amino acids may be substituted with other amino acids in a protein or polypeptide sequence without significantly impairing or impairing the interactive binding ability with structures such as the antigen-binding region of an antibody or the binding site on a substrate molecule. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in the protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Thus, the inventors contemplate that various changes may be made in the DNA sequence of a gene encoding a protein without significantly impairing its biological usefulness or activity.

[0202] As used herein, the term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in a codon that code for a biologically equivalent amino acid.

[0203] The variants of the amino acid sequences of the present disclosure can be substitution variants, insertion variants, or deletion variants. Mutations in the polypeptides of the present disclosure can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide compared to the wild type. Variants can include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical (including all values ​​and ranges therebetween) to any of the sequences provided or referenced herein. The variant can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0204] It will also be understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, respectively, and still be essentially identical to that set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including maintenance of biological protein activity with respect to protein expression. The addition of terminal sequences applies particularly to nucleic acid sequences, which may include various non-coding sequences, for example adjacent to either the 5' or 3' portion of the coding region.

[0205] Deletion mutants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted, or a number of consecutive amino acids can be deleted. Stop codons can be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate truncated proteins.

[0206] Insertional mutants typically involve the addition of an amino acid residue at a non-terminal point of the polypeptide. This may involve the insertion of one or more amino acid residues. Terminal additions may also be produced and may include fusion proteins that are multimers or concatamers of one or more of the peptides or polypeptides described or referenced herein.

[0207] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide and may be designed to modulate one or more properties of the polypeptide, with or without impairing other functions or properties. Substitutions may be conservative, i.e., one amino acid is replaced with an amino acid having similar chemical properties. "Conservative amino acid substitutions" may involve the exchange of a member of one amino acid class for another member of the same class. Conservative substitutions are well known in the art and include, for example, alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine, or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine. Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics or other reverse or inverted forms of amino acid moieties.

[0208] Alternatively, substitutions may be "non-conservative" and thus affect the function or activity of the polypeptide. Non-conservative changes typically involve replacing an amino acid residue with a chemically different amino acid residue, e.g., substituting a polar or charged amino acid for a non-polar or uncharged amino acid and vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0209] 2. Substitution Considerations Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. Those skilled in the art can identify suitable regions of the molecule that can be altered without impairing activity by targeting regions that are not believed to be important for activity. Those skilled in the art can also identify amino acid residues and parts of molecules that are conserved between similar proteins or polypeptides. In further embodiments, regions that may be important for biological activity or structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or adversely affecting the structure of the protein or polypeptide.

[0210] In making such changes, the hydropathic index of amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​iteratively along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. These are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cysteine ​​(+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). The importance of the hydropathic amino acid index in conferring biological interactive functions to a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein or polypeptide, which in turn dictates the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids may be substituted with other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on hydropathic index, certain embodiments include substitutions of amino acids with hydropathic indexes within ±2. Some aspects of the invention include those within ±1, and other aspects of the invention include those within ±0.5.

[0211] It is also understood in the art that substitution of similar amino acids can be effectively performed based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the greatest local average value of the hydrophilicity of a protein, as determined by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. In certain embodiments, the greatest local average value of the hydrophilicity of a protein, as determined by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding, i.e., the biological properties of the protein. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1), glutamic acid (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5±1), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, certain embodiments include substitutions of amino acids with hydrophilicity values ​​within ±2, other embodiments include substitutions of amino acids with hydrophilicity values ​​within ±1, and still other embodiments include substitutions of amino acids with hydrophilicity values ​​within ±0.5. In some cases, epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid with a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0212] Additionally, one of skill in the art can review structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues in the similar protein that are important for activity or structure. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0213] The skilled artisan can also analyze the three-dimensional structure of similar proteins or polypeptides and the amino acid sequence relative to that structure. In view of such information, the skilled artisan may predict the alignment of the amino acid residues of the antibody relative to the three-dimensional structure. The skilled artisan may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. In addition, the skilled artisan may generate test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using standard assays of binding and / or activity, and the information gathered from such routine experiments will allow the skilled artisan to determine the amino acid positions where further substitutions need to be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the world wide web at expasy.org / proteomics / protein_structure.

[0214] In some embodiments of the invention, amino acid substitutions are made that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in naturally occurring sequences. Substitutions can be made in portions of antibodies that are located outside of the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially change the structural features of the protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes the native antibody) can be used.

[0215] V. Nucleic acids In certain embodiments, nucleic acid sequences can be present in various forms, such as isolated segments of integrated sequences or recombinant polynucleotides and recombinant vectors that encode the peptides and polypeptides of the present disclosure or fragments, derivatives, muteins, or variants thereof, polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify polynucleotides encoding the polypeptides, antisense nucleic acids to inhibit expression of the polynucleotides, and complementary sequences as previously described herein. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0216] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from whole genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors (including, for example, plasmids, cosmids, phages, viruses, etc.). A polynucleotide, in certain embodiments, includes a regulatory sequence that is substantially isolated from a naturally occurring gene or protein-coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0217] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences necessary for correct transcription, post-translational modification, or localization). As will be understood by those skilled in the art, the term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide may include a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that includes variants having slightly different nucleic acid sequences, but still encode the same or substantially similar protein.

[0218] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein, which variants comprise at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity (including all values ​​and ranges therebetween) compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% or more identity over the entire length of the sequence to the amino acid sequence described herein, or a nucleotide sequence complementary to the isolated polynucleotide described above.

[0219] Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., such that the overall length may vary significantly. The nucleic acid may be of any length. It may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, or more nucleotides in length, and / or may include one or more additional sequences, e.g., regulatory sequences, and / or may be part of a larger nucleic acid, e.g., a vector. Thus, it is contemplated that a nucleic acid fragment of almost any length may be used, although the total length is preferably limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides may be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0220] A. Hybridization A nucleic acid that hybridizes with another nucleic acid under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a hybridization buffer of 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide, a pre-wash solution of 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing about 50% formamide at a hybridization temperature of 42°C), and washing conditions of 0.5x SSC, 0.1% SDS at 60°C. Stringent hybridization conditions are hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids containing nucleotide sequences that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to each other will typically remain hybridized to each other.

[0221] Parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are provided, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated by reference in their entireties for all purposes, and can be readily determined by one of skill in the art, based, for example, on the length and / or base composition of the DNA.

[0222] B. Mutation Changes can be introduced into a nucleic acid by mutation, thereby resulting in a change in the amino acid sequence of a polypeptide (e.g., an antigenic peptide or polypeptide) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, e.g., using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, e.g., using a random mutagenesis protocol. However made, the mutant polypeptides can be expressed and screened for desired properties.

[0223] Mutations can be introduced into a nucleic acid without causing a significant change in the biological activity of the polypeptide that the nucleic acid encodes. For example, nucleotide substitutions can be made to cause amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively change the biological activity of the polypeptide that the nucleic acid encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutations can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increased, decreased, or lost activity. Examples of qualitative changes include changes in the antigen specificity of an antibody.

[0224] C. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for detection of a nucleic acid sequence. A nucleic acid molecule can include only a portion of a nucleic acid sequence encoding a full-length polypeptide (e.g., a fragment that can be used as a probe or primer, or a fragment that encodes an active portion of a given polypeptide).

[0225] In another embodiment, the nucleic acid molecule may be used as a probe or PCR primer for a specific nucleic acid sequence. For example, the nucleic acid molecule probe may be used in diagnostic methods, and the nucleic acid molecule PCR primer may be used to amplify a region of DNA that can be used to isolate a nucleic acid sequence, particularly for use in generating the engineered cells of the present disclosure. In a preferred embodiment, the nucleic acid molecule is an oligonucleotide.

[0226] Probes based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or similar nucleic acids, such as transcripts encoding the polypeptide of interest. The probes can contain a label group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells that express the polypeptide.

[0227] VI. Polypeptide Expression In some embodiments, there is a nucleic acid molecule encoding the polypeptide, antibody, or antigen-binding fragment of the present disclosure. The nucleic acid molecule can be used to express large amounts of the polypeptide. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule can be used to humanize the antibody or TCR gene.

[0228] A. Vector In some embodiments, expression vectors comprising a nucleic acid molecule encoding a polypeptide of desired sequence or a portion thereof (e.g., a fragment comprising one or more CDRs or one or more variable region domains) are contemplated. The expression vector comprising the nucleic acid molecule may encode a heavy chain, a light chain, or an antigen-binding portion thereof. In some embodiments, the expression vector comprising the nucleic acid molecule may encode a fusion protein, a modified antibody, an antibody heavy and / or light chain, an antibody fragment, and a probe thereof. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that perform other functions as well.

[0229] To express a polypeptide or peptide of the invention, DNA encoding the polypeptide or peptide is inserted into an expression vector such that the gene region is operably linked to transcriptional and translational control sequences. In some embodiments, the vector encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In some embodiments, the vector encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered so that any variable sequence or CDR1, CDR2, and / or CDR3 can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or viral maintenance, as well as sequences for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Such sequences and methods for their use are well known in the art.

[0230] B. Expression Systems There are many expression systems that include at least some or all of the above expression vectors. Prokaryotic and / or eukaryotic based systems can be used in the embodiments to produce the nucleic acid sequence or its cognate polypeptide, protein, and peptide. Commercially widely available systems include, but are not limited to, bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms of action for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. Those skilled in the art can use appropriate expression systems to express the vectors that produce the nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0231] C. Methods of gene transfer Suitable methods for nucleic acid delivery to effect expression of the compositions are contemplated to include virtually any method by which a nucleic acid (e.g., DNA (including viral and non-viral vectors)) can be introduced into a cell, tissue, or organism, as described herein or known to one of skill in the art. Such methods include direct delivery of DNA, e.g., by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, each of which is incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, each of which is incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al. al., 1990); use of DEAE-dextran followed by polyethylene glycol (Gopal, 1985); direct sonic loading (Fechheimer et al., 1987); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1990). al., 1991); microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and WO 95 / 06128; U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each of which is incorporated herein by reference); stirring with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); delivery via desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include gene transfer by viral transduction, e.g., lentiviral or retroviral transduction.

[0232] VII. Cancer Treatment In some aspects, the methods of the disclosure include administering a cancer treatment to the subject or patient. The cancer treatment may be selected based on the expression level measurement, alone or in combination with a clinical risk score calculated for the subject. The cancer treatment may be selected based on the subject's genotype. The cancer treatment may be selected based on the presence or absence of one or more polymorphisms in the subject. In some aspects, the cancer treatment includes a local cancer treatment. In some aspects, the cancer treatment excludes a systemic cancer treatment. In some aspects, the cancer treatment excludes a local treatment. In some aspects, the cancer treatment includes a local cancer treatment without administering a systemic cancer treatment. In some aspects, the cancer treatment includes an immunotherapy, which may be a checkpoint inhibitor treatment. Any of these cancer treatments may be excluded. A combination of these treatments may be administered.

[0233] As used herein, the term "cancer" may be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain aspects, the cancer may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some aspects, the cancer is a stage I cancer. In some aspects, the cancer is a stage II cancer. In some aspects, the cancer is a stage III cancer. In some aspects, the cancer is a stage IV cancer.

[0234] The cancer may specifically be of the following tissue types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma. carcinoma);adrenal cortical carcinoma;endometrioid carcinoma;adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;earwax gland carcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant ;membranoma, malignant;granulosa cell tumor, malignant;astroblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomus angiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma of giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;mucin Liquid sarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stroma sarcoma;Mixed tumor, malignant;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Epithelioid hemangioendothelioma , malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma, malignant;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;oligodendroglioma; oligodendroglioblastoma; primitive neuroectodermal neuroectodermal);cerebellar sarcoma;ganglionioblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease;Hodgkin's paragranuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphocytic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0235] In some embodiments, the cancer is an aggressive cancer. In some embodiments, the cancer is a stage I cancer. In some embodiments, the cancer is a stage II (e.g., IIA, IIB, IIC) cancer. In some embodiments, the cancer is a stage III (e.g., IIIA, IIIB, IIIC) cancer. In some embodiments, the cancer is a stage IV (e.g., IVA, IVB) cancer.

[0236] The methods may include determining, managing, or selecting an appropriate cancer "management regimen" and predicting its outcome. As used herein, the phrase "management regimen" refers to a management plan that prescribes the type of testing, screening, diagnosis, monitoring, care, and treatment (e.g., dosage, treatment schedule, and / or duration) to be provided to a subject in need thereof (e.g., a subject diagnosed with cancer).

[0237] A. GHR Targeting Polypeptides Aspects of the present disclosure include administering one or more GHR targeting polypeptides of the present disclosure to a subject. A subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GHR targeting polypeptides of the present disclosure. A GHR targeting polypeptide of the present disclosure may be administered alone or in combination with any other cancer treatment known in the art and / or described herein. A GHR targeting polypeptide of the present disclosure may be administered to a subject in a variety of forms, including as a recombinant polypeptide, as a nucleic acid encoding the polypeptide, and as a cell that expresses or is capable of expressing the polypeptide (e.g., an immune cell such as a T cell or an NK cell).

[0238] B. Combination Therapy In some aspects, the methods may further comprise administering an additional therapy, such as radiation therapy, cancer immunotherapy, an oncolytic virus, a chemotherapeutic agent, a hormonal therapy, or surgery. Specific chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines, and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin), and triazines (e.g., dicarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine), and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipox, and the like. dophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) various drugs, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxybutyric acid, urea), methylhydiazine derivatives (e.g., procarbazine), and adrenal cortical suppressants (e.g., taxol and mitotane), anti-microtubule agents such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"), pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil, 5-FU), and floxuridine (fluorodeoxyuridine, FudR).

[0239] VIII. Treatment of Acromegaly In some aspects, the methods of the disclosure include administering to the subject or patient a treatment for acromegaly. In some aspects, the treatment for acromegaly is surgery (e.g., removal of a pituitary adenoma). In some aspects, the treatment for acromegaly is radiation therapy. In some aspects, the treatment for acromegaly includes treatment with a therapeutic agent. In some aspects, the therapeutic agent is a somatostatin analog (e.g., octreotide, lanreotide). In some aspects, the therapeutic agent is a dopamine receptor agonist (e.g., cabergoline). In some aspects, the therapeutic agent is a GHR antagonist (e.g., pegvisomant). In some aspects, the therapeutic agent is a GHR targeting polypeptide of the disclosure (e.g., an antibody or antibody fragment that specifically binds to GHR). Any of these acromegaly treatments may be excluded. A combination of these treatments may be administered.

[0240] A. GHR Targeting Polypeptides Aspects of the present disclosure include administering to a subject one or more GHR-targeting polypeptides of the present disclosure (e.g., GHR-targeting antibodies, GHR-targeting antibody fragments, etc.). A subject may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GHR-targeting polypeptides of the present disclosure. GHR-targeting polypeptides of the present invention may be administered alone or in combination with any other acromegaly treatments known in the art and / or described herein. GHR-targeting polypeptides of the present invention may be administered to a subject in a variety of forms, including as recombinant polypeptides, as nucleic acids encoding the polypeptides, and as cells that express or are capable of expressing the polypeptides (e.g., immune cells such as T cells or NK cells).

[0241] IX. Pharmaceutical Compositions The present disclosure includes methods of treating disease and modulating an immune response in a subject in need thereof. The present disclosure includes cells, which may be in the form of a pharmaceutical composition, that can be used to induce or modify an immune response.

[0242] Administration of compositions according to the present disclosure may typically occur via any common route, including, but not limited to, parenteral, orthotopic, intradermal, subcutaneous, oral, transdermal, intramuscular, intraperitoneal, intraperitoneally, intraorbital, implant, inhalation, intraventricular, intranasal, or intravenous injection. In some embodiments, a composition of the present disclosure (e.g., a composition comprising a GHR binding polypeptide) is administered to a subject intravenously.

[0243] Typically, compositions and treatments of this disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunomodulating. The amount administered will depend on the subject being treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0244] The mode of application may be very varied. Any of the conventional methods for administering pharmaceutical compositions containing cellular components are applicable. The dosage of the pharmaceutical composition depends on the route of administration and varies according to the size and health of the subject.

[0245] In many cases, multiple doses, up to 3, 4, 5, 6, 7, 8, 9, 10 or more, are desirable. Doses may be spaced from 2 days to 12 weeks apart, but are more usually spaced 1 to 2 weeks apart.

[0246] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not cause adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. The pharmaceutical compositions of the present disclosure are pharma-ceutically acceptable compositions.

[0247] The compositions of the present invention can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions, and the preparations can also be emulsified.

[0248] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0249] Sterile injectable solutions are prepared by incorporating the active ingredient (e.g., a polypeptide of the present disclosure) in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the other required ingredients as enumerated above.

[0250] The effective amount of the composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response as described herein in conjunction with its administration, i.e., the appropriate route and regimen. The amount administered depends on the desired outcome and protection, both depending on the number of treatments and the unit dose. The exact amount of the formulation also depends on the physician's judgment and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the subject, the route of administration, the intended therapeutic goal (alleviation of symptoms or cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, the solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective. The formulation is easily administered in a variety of dosage forms, such as the types of injection solutions described above.

[0251] The compositions and related methods of the present disclosure, and in particular the administration of the compositions of the present disclosure, may also be used in combination with the administration of additional therapeutic agents, such as those described herein, or in combination with other conventional therapeutic agents known in the art.

[0252] The therapeutic compositions and treatments disclosed herein may precede, be contemporaneous with, and / or be followed by another treatment or agent at intervals ranging from minutes to weeks. In embodiments in which the agents are applied separately to a cell, tissue, or organism, it will generally be ensured that no significant period of time passes between the times of each delivery, so that the therapeutic agents can still exert their beneficial combined effect on the cell, tissue, or organism. For example, in such cases, it is contemplated to contact a cell, tissue, or organism with two, three, four, or more agents or treatments substantially simultaneously (i.e., within less than about one minute). In other embodiments, one or more therapeutic agents or treatments are administered 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 9 It may be administered or provided within 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and / or within any range derivable therein.

[0253] The treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered and the particular route and formulation are within the skill of one of ordinary skill in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0254] The dosage depends on the desired therapeutic effect, both in terms of number of treatments and unit dosage. It is understood that an effective amount refers to the amount required to achieve a particular effect. In the implementation of certain embodiments, it is contemplated that a dosage ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, it is contemplated that doses include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times during the day and / or over multiple days, weeks, or months.

[0255] In some embodiments, a therapeutically effective or sufficient amount of an immune checkpoint inhibitor, such as an antibody and / or a microbial regulator, administered to a human ranges from about 0.01 to about 50 mg per kg of patient body weight, whether administered in a single dose or multiple doses. In some embodiments, the therapeutic agent used is administered at, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg per day. In one embodiment, the therapeutic agent described herein is administered to a subject on day 1 of a 21-day cycle at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. The dose may be administered as a single dose or as multiple doses (e.g., two or three doses), such as by infusion. The progress of this treatment is easily monitored by conventional techniques.

[0256] In certain embodiments, an effective dose of the pharmaceutical composition is a dose capable of providing a blood level of about 1 μM to 150 μM. In other embodiments, an effective dose provides a blood level of about 4 μM to 100 μM, or about 1 μM to 100 μM, or about 1 μM to 50 μM, or about 1 μM to 40 μM, or about 1 μM to 30 μM, or about 1 μM to 20 μM, or about 1 μM to 10 μM, or about 10 μM to 150 μM, or about 10 μM to 100 μM, or about 10 μM to 50 μM, or about 25 μM to 150 μM, or about 25 μM to 100 μM, or about 25 μM to 50 μM, or about 50 μM to 150 μM, or about 50 μM to 100 μM (or any range derivable therein).In other embodiments, the dosage will result in the therapeutic agent being administered to a subject at about the following blood levels of the agent: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any derivable range therein, may be provided.In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to become a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that therapeutic agent. Alternatively, the blood levels discussed herein may refer to the unmetabolized therapeutic agent, unless the therapeutic agent is metabolized by the subject.

[0257] The precise amount of a therapeutic composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (alleviation of symptoms or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatments the subject is undergoing.

[0258] It will be understood and appreciated by those skilled in the art that dosage units of μg / kg or mg / kg (body weight) can be converted and expressed in equivalent concentration units of μg / ml or mM (blood levels), such as 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Applicable conversion coefficients and physiological assumptions regarding uptake and concentration measurements are well known and will allow those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding doses, efficacy, and results as described herein.

[0259] X. Detectable Labels In some aspects of the present disclosure, it is useful to detectably or therapeutically label the Fab polypeptide or protein GFab binding domain. Methods for binding polypeptides to these agents are known in the art. By way of example only, the polypeptide can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled polypeptides can be used in diagnostic techniques in vivo or in an isolated test sample, or in the methods described herein.

[0260] As used herein, the term "label" contemplates a directly or indirectly detectable compound or composition that is directly or indirectly attached to a composition to be detected, e.g., a protein such as a polynucleotide or an antibody, to produce a "labeled" composition. The term also includes sequences attached to a polynucleotide that provide a signal, such as green fluorescent protein (GFP), when the inserted sequence is expressed. The label may be detectable by itself (e.g., radioisotope labels and fluorescent labels) or, in the case of enzymatic labels, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for smaller scale detection or may be more suitable for high throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A response that is simply detected generally includes a response whose presence is merely confirmed, whereas a response that is quantified generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response may be generated directly using a luminophore or fluorophore associated with the assay component actually responsible for binding, or may be generated indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0261] Examples of luminescent labels that produce signals include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally includes a change in a luminescent signal or the generation of a luminescent signal. Suitable methods and luminophores for luminescent labeling of assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.thed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0262] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.thed.).

[0263] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of functional group of the fluorescent label depends on the site of attachment to either the linker, drug, marker, or second labeling agent.

[0264] The fluorescent label can be attached directly to the cellular component or compound or via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to an intermediate include, but are not limited to, antigen / polypeptide, e.g., rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0265] The sensitivity of the antibody in the assay can be increased by binding the polypeptide to a low molecular weight hapten. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten polypeptides. See Harlow and Lane (1988), supra.

[0266] XI. Sample Preparation In certain aspects, the method includes obtaining or evaluating a sample from a subject. Samples include samples obtained from any source, including but not limited to blood, sweat, hair follicles, oral tissue, tears, menstrual blood, feces, or saliva. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, may obtain the biological sample for testing. Additionally, the biological sample can be obtained without the assistance of a medical professional.

[0267] Samples include, but are not limited to, cells of a subject or tissues, cells, or biological materials derived from said cells. Biological samples may be heterogeneous or homogeneous cell or tissue populations. Biological samples may be obtained using any method known in the art that can provide a suitable sample for the analytical methods described herein. Samples may be obtained by non-invasive methods, including, but not limited to, skin or cervical scrapings, cheek swabs, saliva collections, urine collections, fecal collections, menstrual blood, tears, or semen collections.

[0268] The sample may be obtained by methods known in the art. In certain embodiments, the sample is obtained by biopsy. In other embodiments, the sample is obtained by swabbing, endoscopy, scraping, bloodletting, or other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of the kit of the method of the present invention. In some cases, multiple samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples may be obtained for diagnosis by the methods, for example, one or more samples from one tissue type (e.g., esophagus) and one or more samples from another specimen (e.g., serum). In some cases, multiple samples may be obtained at the same time or at different times, for example, one or more samples from one tissue type (e.g., esophagus) and one or more samples from another specimen (e.g., serum). Samples may be taken at different times and stored and / or analyzed by different methods. For example, samples may be obtained and analyzed by routine staining or other cytological analysis methods.

[0269] In some embodiments, the biological sample may be obtained by a doctor, nurse, or other medical professional, such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may indicate the appropriate test or analysis to be performed on the sample. In certain aspects, the molecular profiling operator may provide consultation regarding which assay or test is most appropriate. In further aspects of the method, the patient or subject may obtain the biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, an oral sample, or a saliva sample.

[0270] In other cases, samples are obtained by invasive procedures, including but not limited to biopsy, needle aspiration, endoscopy, or phlebotomy. Needle aspiration procedures may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods described herein to ensure a sufficient amount of biological material.

[0271] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is incorporated herein by reference in its entirety, describe general methods for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of a tumor or neoplasm. In some cases, ultrasound, x-ray, or other imaging devices may be used to guide the fine needle aspiration sampling procedure.

[0272] In some embodiments of the methods of the present invention, the molecular profiling enterprise may obtain the biological sample directly from the subject, from a medical professional, from a third party, or from a kit provided by the molecular profiling enterprise or a third party. In some cases, the biological sample may be obtained by the molecular profiling enterprise after the subject, medical professional, or third party obtains the biological sample and sends it to the molecular profiling enterprise. In some cases, the molecular profiling enterprise may provide the molecular profiling enterprise with suitable containers and excipients for storage and transport of the biological sample.

[0273] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. Instead, an individual may obtain a sample using an over-the-counter (OTC) kit. The OTC kit may include means for obtaining said sample as described herein, means for storing the sample for testing, and instructions on the correct use of the kit. In some cases, the purchase price of the kit includes molecular profiling services. In other cases, molecular profiling services are charged separately. A sample suitable for use by a molecular profiling enterprise may be any material that includes tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of the individual to be tested. Methods for determining suitability and / or suitability of a sample are provided.

[0274] In some embodiments, the subject may be referred to a specialist, such as an oncologist, surgeon, or endocrinologist. The specialist may also obtain the biological sample for testing or may refer the individual to a testing center or laboratory to submit a biological sample. In some cases, a medical professional may refer the subject to a testing center or laboratory to submit a biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling entity may obtain the sample.

[0275] XII.Host cells As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms include both freshly isolated cells and cells cultured, activated, or expanded ex vivo. All of these terms also include any and all subsequent generations of progeny. It is understood that all progeny may not be identical due to intentional or unintentional mutations. In the context of expression of heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. Host cells can be, and have been, used as recipients of vectors or viruses. Host cells may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid, such as a sequence encoding a recombinant protein, is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny.

[0276] In certain embodiments, transfection can be performed on any prokaryotic or eukaryotic cell. In some aspects, electroporation involves the transfection of human cells. In other aspects, electroporation involves the transfection of animal cells. In certain aspects, transfection includes the transfection of cell lines or hybrid cell types. In some aspects, the transfected cells are cancer cells, tumor cells, or immortalized cells. In some cases, tumors, cancers, immortalized cells or cell lines are induced, and in other cases, tumors, cancers, immortalized cells or cell lines naturally enter into their respective disease states or conditions. In certain aspects, the cell or cell line is selected from the group consisting of A549, B cells, B16, BHK-21, C2C12, C6, CaCo-2, CAP / , CAP-T, CHO, CHO2, CHO-DG44, CHO-K1, COS-1, Cos-7, CV-1, dendritic cells, DLD-1, embryonic stem (ES) cells or derivatives thereof, H1299, HEK, 293, 293T, 293FT, HepG2, hematopoietic stem cells, HOS, Huh-7, induced pluripotent stem (iPS) cells or derivatives thereof, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, mesenchymal cells, Min-6, monocytic cells, Neuro2a, NIH The cells may be 3T3, NIH3T3L1, K562, NK cells, NS0, Panc-1, PC12, PC-3, peripheral blood cells, plasma cells, primary fibroblasts, RBL, Renca, RLE, SF21, SF9, SH-SY5Y, SK-MES-1, SK-N-SH, SL3, SW403, Stimulus-induced Acquisition of Pluripotency (STAP) cells or derivatives SW403, T cells, THP-1, tumor cells, U2OS, U937, peripheral blood lymphocytes, proliferating T cells, hematopoietic stem cells, or Vero cells.

[0277] XIII. Kit Certain aspects of the present invention also relate to kits that include the compositions of the present disclosure or the compositions for carrying out the methods of the present disclosure. In some embodiments, the kits can be used to detect the presence of cancer cells in a sample. In certain embodiments, the kits include at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any derivable value or range and combination therein. In some embodiments, the kit includes one or more polypeptides capable of binding to GHR, including polypeptides disclosed herein. For example, the kit may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies or antibody fragments (e.g., Fabs) disclosed herein for detecting GHR, in some cases, on the surface of cancer cells. In some embodiments, the kit includes a detection pair. In some embodiments, the kit includes an enzyme. In some embodiments, the kit includes a substrate for the enzyme.

[0278] The kits may be individually packaged or may be contained in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0279] Individual components may also be provided in the kit in concentrated amounts, and in some embodiments, components are provided individually at the same concentration as they are in a solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0280] Kits for using the disclosed probes, synthetic nucleic acids, non-synthetic nucleic acids, and / or inhibitors for prognostic or diagnostic applications are included as part of this disclosure. In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit embodiments.

[0281] The kit may further comprise instructions for use. For example, in some embodiments, the kit comprises instructions for detecting GHR, such as GHR on the surface of cancer cells in a sample.

[0282] Any method or composition described herein can be implemented with respect to any other method or composition described herein, and it is contemplated that the different embodiments may be combined. It is contemplated that the claims as filed can include multiple dependent claims on any filed claim or combination of filed claims. EXAMPLES

[0283] XIV. Examples The following examples are included to demonstrate specific embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0284] Introduction and Methods Using a phage display selection strategy with human growth hormone receptor as the antigen, we generated diverse antibody inhibitors that compete with human growth hormone binding and reduce its downstream signaling. In vitro protein biochemistry, biophysical characterization, and cell-based assays were used to characterize and further develop the synthetic human antibodies. Most Fabs showed high binding affinity and avidity to cells expressing GHR, and one group showed inhibitory activity as determined by reduced STAT5 phosphorylation in IM9 cells. These potent inhibitory antibodies and antibody fragments against GHR may provide new strategies for therapeutic intervention against conditions such as acromegaly, cancer, and longevity.

[0285] Materials and Methods GHR-Fc was constructed by cloning the extracellular region of GHR (amino acids 32-238) using forward primer 5-ctggtgaccaacagcactagtgagcctaaattcaccaagtgc-3 (SEQ ID NO: 490) and reverse primer 5-ccaccaccaccagaactagtttgggacatctgaggaagtg-3 (SEQ ID NO: 491) upstream of the Fc domain (Spel restriction site) in vector pAB1200 (provided by James Wells lab, University of California, San Francisco). The GHR-Fc-10xHis construct was constructed by removing the AviTag in pAB1200 with restriction enzymes BamHI and NheI and inserting oligo 5-gggtggaggcggatcccatcatcatcaccaccaccaccatcatcattaatcccagtgtcgctagctggccagacatg-3 (SEQ ID NO: 492). Full length GHR was cloned into pCMV3 vector using forward primer 5-gtcgctagcgcggccgccaccatggatctctggcagctgctg-3 (SEQ ID NO: 493) and reverse primer 5-ctatgcggccttgaattcttaaggcatgattttgttcag-3 (SEQ ID NO: 494) between restriction sites BamHI and Xhol. Subdomain 2 of GHR (amino acids 128-246) was fused to Fc in vector pAB1200 using forward primer 5-ggcgctggtgaccaacagcactagtatagtgcaaccagatccacccattg-3 (SEQ ID NO: 495) and reverse primer 5-ccaccaccaccagaactagtgtagaaatcttcttcacatgtaaattg-3 (SEQ ID NO: 496) to generate plasmid S2-Fc. Human growth hormone and mutant B2036 were synthesized as described by Kim et al. 24The B2036 mutant was cloned into a pET28 vector with a 6xHis tag upstream, similar to that described by et al. Briefly, the pET28 vector was digested using NheI and XhoI restriction enzymes, and the PCR fragment was inserted using forward primer 5-cagccatatggctagcatgttcccaactataccacta-3 (SEQ ID NO: 497) and reverse primer 5-ggtggtggtgctcgagctagaatccacagctgcc-3 (SEQ ID NO: 498). The sequence used for the B2036 mutant was taken from U.S. Patent No. 5,849,535, which is incorporated herein by reference in its entirety. All cloning was performed by In-Fusion cloning (TAKARA, Cat. No. 639650).

[0286] Fab and IgG cloning Briefly, the CDR-containing regions were cloned into pSFV4 or pRH2.2 vectors by infusion cloning. The N31D mutation was introduced before converting FAB to IgG format to avoid glycosylation near CDR-H2 during IgG expression in human cells. For IgG cloning, the variable region from the light chain was PCR-generated using forward primer 5-ttacgttcgtcgcggccgcagtcgcctccgatatccagatgacccagtc-3 (SEQ ID NO: 499) and reverse primer 5-atggtgcagccaccgtacgtttgatctccaccttggtaccc-3 (SEQ ID NO: 500). The PCR product was cloned into pscsta vector digested with noti and bsiwi enzymes, containing the kappa light chain constant region. The heavy chain was PCR generated using forward primer 5-ttacgttcgtcgcggccgcagtcgccgagatctccgaggttcagctg-3 (SEQ ID NO: 501) and reverse primer 5- tgggcccttggtgctagccgaggagacggtgaccagggttc-3 (SEQ ID NO: 502). The PCR product from the heavy chain was cloned into pSCSTa vector using Noti and Nhel enzymes and the heavy chain was cloned into pSCSTa vector containing Fc domain from human IgG1 scaffold. Cloning was performed by In-Fusion cloning and positive cloning was verified by DNA sequencing. Anti-MBP FAB has been previously described. 25 .

[0287] Fab and IgG purification Fabs were cloned into IPTG-inducible vectors (pRH2.2 or pSFV4) for bacterial expression (E. coli BE21-Gold). Cells were grown for 24 hours at 30°C in TB autoinduction medium (Fisher Scientific; BP9728-5) supplemented with 0.01% glucose, 0.02% lactose, 1.25 mM MgSO4, and 0.4% glycerol. Cells were harvested by centrifugation and sonicated in 50 mM Tris (pH=7.5), 200 mM NaCl, 2 mM PMSF. Lysates cleared by centrifugation were heated at 65°C for 30 minutes. Lysates were then centrifuged at 38,400g for 30 minutes to remove precipitated proteins. Lysates were filtered through a 0.2 pm filter and purified by AKTA purification apparatus equipped with Protein L resin (Cytiva, catalog number 17547815). Peak fractions were then loaded onto an ion-exchange Resource S 1 ml column (GE Healthcare). After washing with 50 mM sodium acetate (pH 5.0), Fab was eluted with a 0% to 100% linear gradient of a buffer containing 50 mM sodium acetate (pH 5.0) and 2 M sodium chloride. Fractions containing pure Fab were pooled, neutralized with 50 mM HEPES (pH 7.5), dialyzed against a buffer containing 50 mM HEPES (pH 7.5) and 200 mM sodium chloride or 1× PBS, concentrated, and stored in aliquots at −80°C. For IgG expression and purification, Expi293 cells (2× l0 in 10 mL) in 125 mL flasks (Coming, Cat. No. 431143) were cultured with 100 mL of 100 mM glycerol (100 mM NaCl) and 100 mM glycerol (100 mM NaCl). 6Cells / mL) were transfected with 7.5 μg each of light (kappa scaffold) and heavy (IgG1 scaffold) chains in pSCTa plasmid using Opti-MEM medium (Gibco, Cat. No. 31985070) and FectoPro transfection reagent (Polyplus, Cat. No. 116-010) according to the manufacturer's recommendations. The supernatant was then collected 5 days after transfection, remaining cells were removed by centrifugation, and IgG was purified by Protein A resin (GE Healthcare, Cat. No. 17127903) followed by elution with glycine. The buffer was then exchanged into 1×PBS and the IgG was stored at 4° C. (several months) or frozen at −80° C. for longer term storage.

[0288] Purification of human growth hormone and its receptor Human growth hormone and the B2036 variant were purified as previously described. 24Briefly, hormones in pET28 vectors were introduced into E. coli BL21(DE3) cells, grown in LB medium, and induced with IPTG (0.5 mM) for 16 h at 16 °C, and cells were pelleted by centrifugation and lysed by sonication in buffer (50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.1% Triton X-100). Lysates were then purified by Ni-NTA agarose resin (Qiagen, Cat. No. 30250), followed by size-exclusion chromatography on a HiLoad26 / 30 Superdex 200 column (GE Healthcare). Proteins were dialyzed and frozen at -80 °C. Human growth hormone receptor and subdomain 2 (S2) constructs fused to the Fc domain were expressed in Expi293 cells (ThermoFisher, A14527) by transient transfection using FectoPro transfection reagent (Polyplus, Cat. No. 116-010), cells were maintained for 6 days before media harvest and then purified by protein A affinity chromatography using the AKTA purification system. Proteins were biotinylated in vitro using a commercially available kit (AVIDITY, Cat. No. BirA500) according to the manufacturer's recommendations.

[0289] cell line HEK-GHR cell line was generated by transfecting HEK293 cells with pCMV3-GHR plasmid using Lipofectamine LTX reagent. Cells were grown in G418 antibiotic medium for one week for selection, followed by cell sorting for higher expression. HEK-GHR cell line was grown in DMEM medium (10% FBS, 1% penicillin / streptomycin, 0.5mg / mL G418). IM9 cells (Cat. No. CCL-159) and MDAMB435S (Cat. No. HTB-129™) were purchased from ATCC and grown in RPMI medium (10% FBS, 1% penicillin / streptomycin).

[0290] Phage display selection Phage display selection was performed as described above. 26、27 Briefly, biotinylated GHR-Fc was immobilized on streptavidin-coated paramagnetic beads (Promega, Z5482) for four rounds of phage selection. In the first round, 100 nM of target was immobilized on 200 pL of beads and mixed with 1 mL of phage library (10 × 10) with gentle shaking. 11 cfu) at room temperature for 1 hour. The beads were washed three times with 2% BSA-PBST buffer and then used to infect mid-logarithmic phase E. coli XL-1 blue cells (Stratagene) for 20 minutes. They were then incubated with 100 μg / mL ampicillin and 10× 9 Media containing pfu / mL M13K07 helper phage (NEB) was added and phages were grown overnight at 37°C. For the next round, phages were precipitated in 20% PEG / 2.5M NaCl on ice for 20 min. Before each round, precipitated phages were negatively selected against paramagnetic beads containing bound hGHR-hGH complexes for 30 min with shaking to remove binders to the hGHR-hGH complexes. The buffer used for selection was spiked with 200 nM of non-biotinylated competitors (CD8a and CD4 immunoreceptors fused to Fc) to reduce non-specific binders and Fc domain binders. Target concentrations for the next round were 50 nM for the second round, 10 nM for the third round, and 5 nM for the fourth round. During each round, phages were eluted with 0.1 M glycine (pH 2.6) and neutralized with Tris-HCl (pH 8). Phages were then eluted and used for XE-1 infection and phage propagation overnight. Additional phage display selections were performed to generate binders to domain S2 of GHR or to different epitopes in domain 1 (masking selection using FAB4). Unique clones and optimal binders were screened by single point phage EEISA.

[0291] Phage and protein ELISA For phage ELISA, biotinylated targets were immobilized at 50 nM on neutravidin-coated plates (96-well plates, GeinerBio, high binding). After blocking with 5% BSA, phages were added and incubated for 15 min, and then wells were washed extensively before incubation with anti-M13 antibody diluted 1:5,000 in PBST (Abeam; catalog no. b50370). For protein ELISA, FAB was added at different concentrations based on the experimental objectives (single-point ELISA vs. multipoint ELISA). Plates were washed and developed with TMB substrate (Thermo Scientific) and quenched with 1 M HCL, followed by measurement of absorbance at 450 nm (FAB signal) and 570 nm (background subtraction). Protein ELISA protocol was performed as described above. 28 For FAB detection, anti-human FAB-HRP antibody (Jackson Immuno Research, Cat. No. 209-035-097) was used at a dilution of 1:500.

[0292] SPR Surface plasmon resonance (SPR) measurements were performed on a MASS-1 (Bruker) instrument. The target GHR-Fc (50 nM) with 10xHis tag was captured on a Ni-NTA sensor chip. To determine affinity, FABs were run starting at 200 nM followed by 2-fold dilutions (8-fold dilutions). FABs with very high affinity were run starting at 50 nM instead of 200 nM. The flow rate was 30 μl / min and the temperature was 20°C. Sensorgrams were double-referenced and fitted with a 1:1 binding model using a Sierra Analyzer (Bruker). For epitope binning experiments, the first FAB was injected at saturating concentration (200 nM) followed by the second FAB in an equimolar mixture with the first FAB and response units (RU) were recorded.

[0293] Flow cytometry To determine the binding of FAB to GHR in different cell lines (HEK293-GHR and MDAMB435S), cells were trypsinized from the plate using TryLE (ThermoFisher, Cat. No. 12604013) in 1x PBS and incubated at 0.3 x 10 per well in a 96-well plate (Greinerbio-one, Cat. No. 650261). 6 Cells were plated on 100 wells in a 10-well plate. Cells were stained with FAB (200 nM) for 30 min at 4 °C. FAB was detected by a secondary antibody conjugated to Alexa647 (Jackson immunoresearch, Cat. No. 109-605-006). To measure the phosphorylation levels of endogenous STAT5 by flow cytometry, we followed a published method with some modifications. 29 IM9 cells were serum-starved overnight (16 h) in serum-free medium (RPMI, 2x L-glutamine, 1x sodium pyruvate, 1x non-essential amino acids, 1x penicillin / streptomycin). The next day, cells were incubated with 200 nM FAB (50 μL per well) or serial dilutions (IC50 determination). Cells were then incubated with hGH (35 nM) at 37°C, 5% CO 2 The cells were stimulated with 4% PFA in 1× PBS for 10 min at room temperature, washed with 1× PBS, permeabilized with 90% methanol for 30 min on ice, washed twice, and then incubated overnight with a mixture of STAT5-pY694 antibody (Cell Signaling, Cat. No. 9351S) and STAT5-pY694 (Cell Signaling, Cat. No. 9365S) (60:40 ratio) at a 230:1 dilution (50 μL per well). The next day, the cells were washed and incubated with a secondary anti-rabbit antibody conjugated with Alexa-647 (Biolegend, poly4064, Cat. No. 406414) for 30 min at 4° C., fixed, and read by flow cytometry (CytoFlex instrument; Beckman Coulter).

[0294] GHR_TS23 soft randomization A phage library of GHR_TS23 for soft randomization with affinity maturation was generated using a previously published strategy 1. For that purpose, a stop codon was introduced into CDR-H3 by quick-change mutagenesis. A soft randomization strategy was used with phosphorylated oligos, where 50% wild type or 50% all other amino acids were introduced at each position, depending on the library design. ssDNA containing the stop codon introduced in the middle of CDR-H3 was isolated from the phage (using QIAprep Spin M13 Kit, Qiagen) and used in the Kunkel mutagenesis protocol 2. The Kunkel reaction was purified (using Wizard SV Gel and PCR Clean-Up System, Promega) and electroporated into TGI cells (Lucigen). After 1 h recovery at 37 °C with shaking, 40 mL of 2xYT medium (supplemented with 100 μg / mL ampicillin and helper phage) was added to start phage production. The next day, the library was precipitated in 20% PEG / 2.5M NaCl on ice for 20 min. Three rounds of biopanning were performed with different target concentrations (1st round: 10 nM, 2nd round: 1 nM, 3rd round: 100 pM). Phage were then sequenced and independent clones were reformatted into the RH2.2 expression vector and generated for further validation.

[0295] Competitive ELISA To evaluate the inhibitory efficiency of the Fabs, ELISA was used to test the competition with GH for GHR binding. 200 nM GHR was immobilized on a high-binding plate, followed by extensive blocking with BSA, and then incubation with 200 nM GH for 30 min. Each Fab was then added at 12 concentrations (starting at 1 μM and then diluted 2-fold), incubated for 15 min, followed by three extensive washes. Binding of the Fabs to the GHR was detected using an HRP-conjugated anti-human (Fab)2 antibody (Jackson ImmunoResearch, 1:5000 dilution in PBST). The signal was then developed with TMB substrate (Thermo Scientific) and quenched with 10% H3PO4, followed by measuring absorbance at A450. Using GraphPad Prism, the data were fitted using a standard dose-dependent binding model to obtain the EC 50 values ​​were calculated.

[0296] Example 1 - Development of a synthetic human monoclonal antibody that inhibits growth hormone receptor signaling GHR_Fc was expressed in mammalian cells as described below and biotinylated using Avi-tag and BirA enzyme. Four rounds of selection were performed on a high diversity (1010) synthetic library (Miller et al., 2012) using previously published protocols (Fellouse et al., 2007; Paduch et al., 2013). To obtain high affinity binders, the concentration of antigen was stepwise decreased starting from 100 nM in the first round to 5 nM in the fourth round. Furthermore, phages were negatively selected against the GHR-GH complex for 30 min to obtain binders targeting the GH binding site. Phage ELISA was performed on 192 clones, identifying 16 unique high affinity variants (GHR1-GHR16). Protein ELISA revealed two populations of binders: GHR1-GHR13, which may bind to the S1 domain of GHR, and GHR14-GHR16, which exclusively bind to the GHRS2 domain. The Fabs were further characterized and their binding affinities determined by surface plasmon resonance (SPR) (Table 4). All antibodies show low nM binding constants (KD) and slow dissociation rates.

[0297] An important component in developing therapeutics includes the ability to test in species other than primates. To ensure cross-reactive binding of antibodies to GHR across species, additional phage display selections were performed. To that end, five rounds of selection were performed, stepwise decreasing the antigen concentration starting from 500 nM in the first round and ending at 1 nM in the fifth round. GHR_Fc from different species were used as targets in subsequent selection rounds (first round: human, second round: pig, third round: rabbit, fourth round: human, fifth round: human). Furthermore, as mentioned above, phages were negatively selected against the GHR-GH complex for 30 min before each selection round to obtain binders targeting the GH binding site. Phage ELISA was performed on 192 clones, and 44 unique high-affinity variants (GHR_TS1–GHR_TS44) were identified (Figure 4).

[0298] The binding kinetics of the generated Fabs were analyzed by signal plasmon resonance (SPR). Table 4 provides the results of the SPR analysis. [Table 4]

[0299] [Table 5]

[0300] Example 2 - Generation of Fab O.D. 600 After induction with 1 mM IPTG at pH = 0.8–1, Fab was expressed in the periplasm of E. coli BL21 cells at 37 °C for 4 h. Cells were harvested by sonication. After centrifugation, the supernatant was applied to a Protein L affinity column (Cytiva). Protein was eluted from the column with 0.1 M glycine (pH 2.6), neutralized with 1 M Tris-HCl (pH 8.5), and dialyzed overnight into PBS.

[0301] Example 3 - Production of IgG Full-length IgG was incubated at 2 × 10 in Expi293 cells. 6 Expression was at 1000 ng / mL. Heavy chain (IgG1 scaffold) and light chain (kappa) were co-transfected using FectoPro transfection reagent (Polyplus) according to the manufacturer's recommendations. Five days after transfection, the supernatant was collected by centrifugation and IgG was purified using Protein A resin (Cytiva). Protein was eluted from the column with 0.1 M glycine (pH 2.6), neutralized with 1 M Tris-HCl (pH 8.5) and dialyzed overnight into PBS.

[0302] Example 4 - GHR Fab Binding by ELISA Assay To verify the binding of GHR Fab to GHR, an ELISA assay was performed. 50 nM biotinylated GHR was immobilized on a 96-well neutravidin-coated plate (Geiner) followed by extensive blocking with BSA. Phage or purified Fab was incubated on the plate for 15 min, washed three times and incubated with anti-M13 phage (Abeam) or anti-human Fab (Jackson Immunoresearch) antibodies conjugated to HRP (diluted 1:5000 in PBST) for 20 min at room temperature. Plates were then washed and developed with TMB substrate (Thermo Scientific) and quenched with 10% H3PO4, followed by absorbance determination at A450.

[0303] Example 5 - GHR Fab binding by surface plasmon resonance analysis All surface plasmon resonance (SPR) analyses were performed on a MASS-1 (Bruker) to determine the binding and dissociation constants of GHR Fabs. Target GHR_Fc (from different species) was immobilized on a Ni-NTA sensor chip via 10xHis-tag. Two-fold dilutions of Fabs were run as analytes at a flow rate of 30 μl / min at 20°C. Sensorgrams were double-referenced and fitted with a 1:1 binding model using a Sierra Analyzer (Bruker). Epitope binning experiments were performed to reveal different epitopes on GHR. After injecting a saturating concentration (200 nM) of the first Fab, an equimolar mixture of the second Fab was injected and an increase in response units (RU) was observed.

[0304] Example 6 - GHR Fab binding to cells expressing GHR. Flow cytometry analysis was performed to determine binding of the Fab to cell lines expressing GHR (MDA-MB-435 and IM9). GHR Fab was incubated with cells for 30 min at 4 °C and detected by an anti-human Fab secondary antibody conjugated to Alexa647 using a flow cytometer (CytoFlex instrument; Beckman Coulter). Significant binding was observed compared to nonspecific antibodies recognizing MBP or the SARS-CoV-2 RBD protein (Slezak and Kossiakoff, 2021) (Figure 3).

[0305] Example 7 - Verification of GHR_TS23 cross-reactivity SPR was used to verify the binding of GHR_TS23 Fab to GHR from different species. GHR_Fc with 10xHis tag from rabbit, rat, mouse, cynomolgus monkey, and pig was expressed in mammalian cells and immobilized on a Ni-NTA sensor chip. The Fab was run in 2-fold serial dilutions starting from 50 nM at a flow rate of 30 μM / min, confirming the broad cross-reactivity of GHR_TS23 Fab (Figure 5).

[0306] Example 8 - Fab competition assay. The efficiency of GHR_TS23 Fab in competing with human growth hormone (hGH) for binding to the growth hormone receptor was measured by ELISA competition assay (Figure 6A). 200 nM GHR was immobilized on a 96-well high-binding plate (GeinerBio) and blocked overnight with 5% BSA. 200 nM human GH was preincubated with the plate for 1 h at room temperature. Then, two-fold serial dilutions of GHR_TS23 were added to the plate and incubated for 15 min, followed by extensive washing three times with PBST / 0.1% BSA. Fab binding to GHR was detected using anti-human (Fab)2 antibody conjugated to HRP (JacksonImmuno Research, 1:5000 dilution in PBST) and signal was developed using TMB substrate. Efficient competition for the GH binding site was observed with a concentration-dependent profile (Figure 6B).

[0307] Example 9 - GH competition assay The ELISA competition assay determines whether human growth hormone (hGH) can compete out GHR_TS23 Fab from the binding site of the growth hormone receptor. 200 nM of GHR was immobilized on a 96-well high-binding plate (GeinerBio) and blocked overnight with 5% BSA. 200 nM of GHR_TS23 Fab was pre-incubated with the plate for 1 h at room temperature. Then, 2-fold serial dilutions of GH were added to the plate and incubated for 15 min, followed by extensive washing three times with PBST / 0.1% BSA. Fab binding to GHR was detected using an anti-human (Fab)2 antibody conjugated to HRP (JacksonImmuno Research, 1:5000 dilution in PBST) and the signal was developed using TMB substrate. The results show that growth hormone cannot compete out GHR_TS23 Fab from its binding site (Figure 6C).

[0308] Example 10 - GH competition assay The ELISA competition assay determines whether B2036 (an affinity-improved GHR antagonist) can compete out GHR_TS23 Fab from the binding site of the growth hormone receptor. 200 nM of GHR was immobilized on a 96-well high-binding plate (GeinerBio) and blocked overnight with 5% BSA. 200 nM of GHR_TS23 Fab was preincubated with the plate for 1 h at room temperature. Then, 2-fold serial dilutions of B2036 were added to the plate and incubated for 15 min, followed by extensive washing three times with PBST / 0.1% BSA. Fab binding to GHR was detected using an anti-human (Fab)2 antibody conjugated to HRP (JacksonImmuno Research, 1:5000 dilution in PBST) and the signal was developed using TMB substrate. The results show that B2036 cannot compete out GHR_TS23 Fab from its binding site (Figure 6D).

[0309] Example 11 - GHR signaling inhibition assay To determine the inhibitory activity of GHR Fab, phosphorylation of endogenous STAT5 was measured by flow cytometry in IM9 cells. IM9 cells express GHR on their surface and have been used previously to study GH-mediated receptor dimerization and signaling (Clayton et al., 1994). The day before the experiment, IM9 cells were serum-starved for 16 h in RPMI medium (supplemented with 2× L-glutamine, 1× sodium pyruvate, 1× non-essential amino acids, and 1× penicillin / streptomycin). To measure GHR Fab inhibitory activity, cells were incubated with 200 nM GHR Fab for 30 min and then stimulated with 200 nM GH for 10 min at 37° C. Cells were then fixed, permeabilized with 4% PFA and 90% methanol, and washed extensively. STAT5 phosphorylation was detected with anti-STAT5-pY694 antibody (Cell Signaling) incubated overnight at 4° C. The next day, cells were washed and staining was measured by flow cytometry (CytoFlex instrument; Beckman Coulter) using an anti-rabbit secondary antibody conjugated to Alexa-647 (Biolegend). To determine the 50% inhibitory concentration (IC50), GHR Fab was incubated in serial dilutions but the staining protocol was not modified.

[0310] Example 12 - Importance of CDR-H3 composition of GHR_TS23 Fab To establish the importance of each amino acid for GHR binding, soft randomization of the CDR-H3 from GHR_TS23 Fab was performed. Phosphorylated oligos were used to generate a phage display library, with library design introducing 50% wild type or 50% all other amino acids at each position (Figure 9A). ssDNA of GHR_TS23 was isolated from the phage (using QIAprep Spin M13 Kit, Qiagen) and used in the Kunkel mutagenesis protocol (Kunkel, 1985). Reactions were purified (using Wizard SV Gel and PCR Clean-Up System, Promega) and electroporated into TGI cells (Lucigen). After recovery at 37°C for 1 h with shaking, medium supplemented with helper phage (NEB) was added to initiate phage production. The following day, phages were precipitated and incorporated into phage display selection against GHR_Fc. Three rounds of biopanning were performed, and the target concentration was gradually decreased in the subsequent rounds to introduce selection pressure (1st round: 10 nM, 2nd round: 1 nM, 3rd round: 100 pM). Phages were then sequenced, and the results showed a wide diversity at position 2 of CDR-H3. Some positions of GHR_TS23 CDR-H3 did not show sequence flexibility (Figure 9B). The introduction of aspartic acid at position 2 of CDR-H3 was then tested by ELISA and SPR, and this introduction showed no interference with GHR binding (Figure 11, Figure 12A). In contrast, the introduction of aspartic acid at the conserved amino acid position abolished GHR binding (Figure 12B).

[0311] Example 13 - Improved developability of GHR_TS23 Fab Introduction of a negative charge has previously been shown to improve antibody developability and production (Schaefer et al., 2016). The wide diversity of amino acid composition at position 2 of the CDR-H3 from GHR_TS23 indicates efficient GHR binding when an aspartic acid was introduced into the CDR-H3 (GHR_TS23.1 Fab). To test the improved developability with GHR_TS23.1 Fab, the Fab was injected onto a 3 mL Superdex200 column (Cytiva) at a flow rate of 0.3 mL / min using the AKTA Pure Protein Purification system (Cytiva). Introduction of an aspartic acid improved the developability and specificity of GHR_TS23.1 Fab compared to GHR_TS23 Fab (Figure 10).

[0312] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention are described with reference to specific embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0313] References The following references, and references cited elsewhere in this specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are expressly incorporated herein by reference. 1. Dehkhoda, F., Lee, C.M.M., Medina, J., and Brooks, A.J. (2018). The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects. Front. Endocrinol. 0. 2. Brooks, A.J., and Waters, M.J. (2010). The growth hormone receptor: mechanism of activation and clinical implications. Nat Rev Endocrinol 6, 515-525. 3. Wells, J.A. (1996). Binding in the growth hormone receptor complex. PNAS 93, 1-6. 4. Brown, R.J., Adams, J.J., Pelekanos, R.A., Wan, Y., McKinstry, W.J., Palethorpe, K., Seeber, R.M., Monks, T.A., Eidne, K.A., Parker, M.W., et al. (2005). Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nature Structural & Molecular Biology 12, 814-821. 5. 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Claims

1. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 that have at least 80% sequence identity with HCDR1, HCR2, and HCR3 from the heavy chain variable region of an antibody clone in Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 that have at least 80% sequence identity with LCDR1, LCDR2, and LCDR3 from the light chain variable region of the same antibody clone in Table 1.

2. 2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of HCDR1, HCDR2, and HCDR3 of a clone in Table 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of LCDR1, LCDR2, and LCDR3 from a light chain variable region of the same clone in Table 1.

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 80% sequence identity with the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 of Table 1, and the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are derived from the same antibody clone.

4. The antibody or antigen-binding fragment of claim 1 or 2, wherein the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences of HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 in Table 1, and the HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 are derived from the same antibody clone.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with the heavy chain variable region of an antibody clone in Table 1, and / or the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with the light chain variable region of the same antibody clone in Table 1.

6. The antibody or antigen-binding fragment of claim 5, wherein the heavy chain variable region comprises the amino acid sequence of the heavy chain variable region of an antibody clone in Table 1 and / or the light chain variable region comprises the amino acid sequence of the same antibody clone in Table 1.

7. 7. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises heavy chain framework regions (HFR) 1, HFR2, HFR3, and HFR4, and light chain framework regions (LFR) 1, LFR2, LFR3, and LFR4, wherein the HFR1, HFR2, HFR3, and HFR4 comprise amino acid sequences having at least 80% sequence identity with HFR1, HFR2, HFR3, and HFR4, respectively, of an antibody clone in Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprise amino acid sequences having at least 80% sequence identity with LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone in Table 1.

8. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the HFR1, HFR2, HFR3, and HFR4 comprise the amino acid sequences of HFR1, HFR2, HFR3, and HFR4, respectively, of the antibody clone in Table 1, and the LFR1, LFR2, LFR3, and LFR4 comprise the amino acid sequences of LFR1, LFR2, LFR3, and LFR4, respectively, of the same antibody clone in Table 1.

9. 9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70% sequence identity to the heavy chain of an antibody clone in Table 1, and the light chain comprising an amino acid sequence having at least 70% sequence identity to the light chain of the same antibody clone in Table 1.

10. 10. The antibody or antigen-binding fragment of claim 9, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of an antibody clone in Table 1, and the light chain comprising the amino acid sequence of the same antibody clone in Table 1.

11. The antibody of any one of claims 1 to 10, wherein the antibody is human, chimeric, or humanized.

12. The antibody or antigen-binding fragment binds to a growth hormone receptor protein at about 10 －６ M / L ~ approx. 10 －１２ M / L K Ｄ The antibody or antigen-binding fragment of any one of claims 1 to 11, which is an antibody or antigen-binding fragment that binds to

13. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody is a neutralizing antibody.

14. 14. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody is a human antibody, a humanized antibody, a recombinant antibody, a chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, a single domain antibody, or a single chain antibody.

15. The antigen-binding fragment may be a single chain variable fragment (scFv), F(ab') ２ 14. The antigen-binding fragment of any one of claims 1 to 13, which is a Fab', Fab, Fv, or rIgG.

16. A polypeptide comprising an antigen-binding fragment according to any one of claims 1 to 15.

17. 17. The polypeptide of claim 16, wherein the polypeptide comprises at least two antigen-binding fragments, each antigen-binding fragment being independently selected from the antigen-binding fragments of any one of claims 1 to 15.

18. 18. The polypeptide of claim 16 or 17, wherein the polypeptide is multivalent.

19. The polypeptide of any one of claims 16 to 18, wherein the polypeptide is bispecific.

20. A composition comprising an antibody or antigen-binding fragment of any one of claims 1 to 15 or a polypeptide of any one of claims 16 to 19.

21. The composition of claim 20 , wherein the composition comprises a pharmaceutical excipient.

22. The composition of claim 20 or 21, wherein the composition further comprises an adjuvant.

23. The composition of any one of claims 20 to 22, wherein the composition is formulated for parenteral, intravenous, subcutaneous, intramuscular, or intranasal administration.

24. The composition of any one of claims 1 to 23, wherein the composition comprises at least two antibodies or antigen-binding fragments.

25. One or more nucleic acids encoding the antibody or antigen-binding fragment of any one of claims 1 to 15 or the polypeptide of claim 19.

26. A nucleic acid encoding an antibody heavy chain, the nucleic acid having at least 70% sequence identity to one of SEQ ID NOs:414-445.

27. A vector comprising the nucleic acid of claim 25 or 26.

28. 28. A host cell comprising a nucleic acid according to claim 25 or 26 or a vector according to claim 27.

29. 29. The host cell of claim 28, wherein the host cell is a human cell, a B cell, a T cell, a Chinese hamster ovary, an NSO mouse myeloma cell, or a PER.C6 cell.

30. 28. A method for producing a cell, comprising introducing a nucleic acid according to claim 25 or 26 or a vector according to claim 27 into the cell.

31. 31. The method of claim 30, further comprising culturing the cell under conditions that allow expression of a polypeptide from the nucleic acid.

32. 32. The method of claim 31 further comprising isolating the expressed polypeptide.

33. 33. The method of any one of claims 30 to 32, wherein the cell is a human cell, a B cell, a T cell, a Chinese hamster ovary cell, an NSO mouse myeloma cell, or a PER.C6 cell.

34. 28. A method for producing a polypeptide, comprising introducing a nucleic acid according to claim 25 or 26 or a vector according to claim 27 into a cell and isolating the polypeptide expressed from the nucleic acid.

35. 35. The method of claim 34, wherein the cell is a human cell, a B cell, a T cell, a Chinese hamster ovary, an NSO mouse myeloma cell, or a PER.C6 cell.

36. 27. A method of treating or preventing cancer in a subject, comprising administering to the subject an antibody or antigen-binding fragment of any one of claims 1 to 15, a polypeptide of any one of claims 16 to 19, a composition of any one of claims 20 to 24, or a host cell of claim 28 or 29.

37. 37. The method of claim 36, wherein the subject is a human subject.

38. 38. The method of claim 36 or 37, wherein the subject has one or more symptoms of cancer.

39. 38. The method of claim 36 or 37, wherein the subject has no symptoms of cancer.

40. The method of any one of claims 36 to 39, wherein the subject has been diagnosed with cancer.

41. 40. The method of any one of claims 36 to 39, wherein the subject has not been diagnosed with cancer.

42. The method of any one of claims 36-41, wherein the subject has previously been treated for cancer.

43. The method of any one of claims 36 to 42, wherein the subject is administered an additional treatment.

44. 44. The method of claim 43, wherein the additional treatment comprises radiation therapy, chemotherapy, or immunotherapy.

45. A method for treating or preventing acromegaly in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment of any one of claims 1 to 15, a polypeptide of any one of claims 16 to 19, a composition of any one of claims 20 to 24, or a host cell of claim 28 or 29.

46. 46. ​​The method of claim 45, wherein the subject is a human subject.

47. 47. The method of claim 45 or 46, wherein the subject has one or more symptoms of acromegaly.

48. 47. The method of claim 45 or 46, wherein the subject does not have one or more symptoms of acromegaly.

49. 47. The method of claim 45 or 46, wherein the subject has been diagnosed with acromegaly.

50. 47. The method of claim 45 or 46, wherein the subject has not been diagnosed with acromegaly.

51. The method of any one of claims 45 to 50, wherein the subject has previously been treated for acromegaly.

52. 52. The method of claim 51, wherein the subject has been refractory to previous treatments.

53. A method of evaluating a sample from a subject, the method comprising contacting a biological sample or an extract thereof from the subject with at least one antibody, antigen-binding fragment, or polypeptide of any one of claims 1 to 19.

54. 54. The method of claim 53, wherein the at least one antibody, antigen-binding fragment, or polypeptide is operably linked to a detectable label.

55. 55. The method of claim 53 or 54, further comprising incubating the antibody, antigen-binding fragment, or polypeptide under conditions that allow the antibody, antigen-binding fragment, or polypeptide to bind to the antigen in the biological sample or extract thereof.

56. 56. The method of any one of claims 53 to 55, further comprising detecting binding of an antigen to the antibody, antigen-binding fragment, or polypeptide.

57. 57. The method of any one of claims 53 to 56, further comprising contacting the biological sample with at least one capture antibody, antigen, or polypeptide.

58. 58. The method of claim 57, wherein the at least one capture antibody, antigen-binding fragment, or polypeptide comprises at least one antibody or antigen-binding fragment of any one of claims 1 to 15.

59. 59. The method of claim 57 or 58, wherein the capture antibody or fragment is linked to a solid support.

60. 60. The method of any one of claims 53 to 59, wherein the biological sample comprises a tissue sample or a blood sample.

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