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psca antibody 7f5  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology psca antibody 7f5
    Discovery and characterization of the <t>PSCA</t> antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody <t>7F5</t> (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.
    Psca Antibody 7f5, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/psca+antibody+7f5/PSCA+Antibody/pmc11312989-26-40-43
    Average 93 stars, based on 30 article reviews
    psca antibody 7f5 - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer"

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    Journal: mAbs

    doi: 10.1080/19420862.2024.2387240

    Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.
    Figure Legend Snippet: Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Flow Cytometry, Control, Concentration Assay, Incubation, FLAG-tag

    Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.
    Figure Legend Snippet: Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

    Techniques Used: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay, Peptide Microarray, Control, Recombinant

    The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Expressing, Fluorescence, Incubation, Comparison, Concentration Assay

    Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.
    Figure Legend Snippet: Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, SDS Page, Enzyme-linked Immunosorbent Assay, Binding Assay, Conjugation Assay, In Vitro, Comparison, Incubation, Cell Viability Assay

    In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: In Vivo, Inhibition, Control, Drug discovery, Injection, Comparison

    Related Articles

    Bacteria:

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer.
    Article Snippet: .. Antibodies, bacteria, cells, and mice The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated antihuman Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRPconjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (SigmaAldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130- 101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514). .. TG1 and DH5a cells were purchased from Lucigen (Cat#60502–1, Cat#60602–1, respectively).



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    Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.

    Article Snippet: The PSCA expression on PC-3 and Du-145 PSCA transgenic cell lines were confirmed by PE-conjugated anti-Flag tag antibody and the murine PSCA antibody 7F5 followed by PE-conjugated anti-mouse IgG antibody (ThermoFisher, CAT#P-852).

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Flow Cytometry, Control, Concentration Assay, Incubation, FLAG-tag

    Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

    Article Snippet: The PSCA expression on PC-3 and Du-145 PSCA transgenic cell lines were confirmed by PE-conjugated anti-Flag tag antibody and the murine PSCA antibody 7F5 followed by PE-conjugated anti-mouse IgG antibody (ThermoFisher, CAT#P-852).

    Techniques: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay, Peptide Microarray, Control, Recombinant

    The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The PSCA expression on PC-3 and Du-145 PSCA transgenic cell lines were confirmed by PE-conjugated anti-Flag tag antibody and the murine PSCA antibody 7F5 followed by PE-conjugated anti-mouse IgG antibody (ThermoFisher, CAT#P-852).

    Techniques: Expressing, Fluorescence, Incubation, Comparison, Concentration Assay

    Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.

    Article Snippet: The PSCA expression on PC-3 and Du-145 PSCA transgenic cell lines were confirmed by PE-conjugated anti-Flag tag antibody and the murine PSCA antibody 7F5 followed by PE-conjugated anti-mouse IgG antibody (ThermoFisher, CAT#P-852).

    Techniques: Liquid Chromatography with Mass Spectroscopy, SDS Page, Enzyme-linked Immunosorbent Assay, Binding Assay, Conjugation Assay, In Vitro, Comparison, Incubation, Cell Viability Assay

    In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The PSCA expression on PC-3 and Du-145 PSCA transgenic cell lines were confirmed by PE-conjugated anti-Flag tag antibody and the murine PSCA antibody 7F5 followed by PE-conjugated anti-mouse IgG antibody (ThermoFisher, CAT#P-852).

    Techniques: In Vivo, Inhibition, Control, Drug discovery, Injection, Comparison

    Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Discovery and characterization of the PSCA antibody fab G7.(a) fab G7 binding to recombinant PSCA-Fc protein measured by ELISA. Bovine serum albumin (BSA) was used as a negative control. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. (b) Kinetics of fab G7 binding to PSCA-Fc, as measured by Blitz. (c) Fab G7 binding to PSCA positive (PC-3-PSCA, Du-145-PSCA and HT1376 cells) and PSCA negative cells (PC-3, Du-145 and CHO-K1 cells) as tested by flow cytometry. An irrelevant fab (anti-SARS-CoV-2 fab ab1) was used as the isotype control. Fab G7 at the concentration of 500 nM was incubated with cells. (d-g) competition of fab G7 and fab F12 binding to HT1376 cell surface-associated PSCA by the recombinant PSCA-Fc protein (d and f) and by the murine PSCA antibody 7F5 (e and g). 500 nM of fab G7 or 200 nM of F12 was incubated with cells in the presence of gradient concentration of competitors. The bound fab G7 or F12 was detected by the pe-conjugated anti-flag tag antibody.

    Article Snippet: The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated anti-human Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRP-conjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (Sigma-Aldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)-conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130-101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514).

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Flow Cytometry, Control, Concentration Assay, Incubation, FLAG-tag

    Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Characterization of the affinity-maturated PSCA antibody F12. (a) the structure model of fab G7, represented as the cartoon model using PyMoL. The light chain and heavy chain CDR3 and FR2 hydrophobic residues are depicted as cyan and red sticks. The orange dotted lines highlight hydrophobic patches in antibody paratopes. (b) Evaluation of binding affinity to PSCA-Fc for affinity-enhanced clones by ELISA. Fab G7 is the parental clone. (c) The kinetics of fab F12 binding to PSCA-Fc, as measured by Blitz. (d) MPA assay to evaluate F12 binding specificity. IgG1 F12 was tested for binding to as many as 6,000 human transmembrane proteins that are transgenically expressed in HEK293 cells in a high throughput manner. (e) Epitope mapping of F12 by the conformational peptide scanning. The PSCA protein was scanned by cyclic peptides with 7, 10 and 13 amino acid length with peptide-peptide overlaps of 6, 9 and 12 amino acids. The conformational PSCA peptide microarray was framed by the HA control peptides. F12 binding was detected by the goat anti-human IgG (H+L) DyLight680 (red color), and the array outmost HA tag peptide was detected by the anti-ha (12CA5) DyLight800 (green color). (f) IgG1 F12 binding to murine PSCA recombinant protein by ELISA. Detection was achieved by hrp-conjugated anti-human fc antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

    Article Snippet: The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated anti-human Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRP-conjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (Sigma-Aldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)-conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130-101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514).

    Techniques: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay, Peptide Microarray, Control, Recombinant

    The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: The internalization of IgG1 F12 into PSCA expressing cancer cells. (a). The scheme of the pH sensitive dye that emits fluorescence only at acidic endosome. (b-d). Evaluation of internalization of IgG1 F12 by using the pH sensitive dye and antibody conjugate. IgG12 F12 was conjugated with pHab dye by the lysine amine-nhs coupling. Different concentrations of F12-pHab were incubated with PSCA+ or – PC-3 and Du-145 cells for 12 or 24 hrs at either 37°C (B and C) or 4°C (d). After washing with 3× PBS, the cell fluorescence was recorded by the fluorescent plate reader, and the signal was normalized to the background. F12 internalization into PC-3 and Du-145 cells was presented in (b) and (c) respectively. (e) Comparison of IgG1 F12 internalization to that of benchmark antibody IgG1 m276, which targets CD276 (B7-H3). Both WT PC-3 and Du-145 cells intrinsically express CD276. (f) The internalization of F12-pHab conjugate can be outcompeted by the naked IgG1 F12 antibody on both PC-3-PSCA and Du-145-psca cells in a concentration dependent manner. The two-way ANOVA followed by Tukey correction was used for statistical analysis. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated anti-human Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRP-conjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (Sigma-Aldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)-conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130-101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514).

    Techniques: Expressing, Fluorescence, Incubation, Comparison, Concentration Assay

    Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: Generation and characterization of ADC IgG1 F12-MMAE. (a) the scheme of ADC IgG1 F12-MMAE. MMAE was conjugated onto IgG1 F12 by the nhs-amine coupling through a cleavable dipeptidyl linker and the PAB spacer. (b) The intact LC/MS spectrum of IgG1 F12-MMAE to determine DAR. The ADC exhibits a heterogenous distribution of DAR with an average DAR of 1.98. (c and d). The quality controls of ADC by SDS-PAGE and ELISA. (c). F12-MMAE and Ab1-mmae HC and LC integrity were checked on SDS-PAGE. (d).The binding to PSCA antigen by F12 was ascertained after conjugation with MMAE. (e and f) the in vitro cytotoxicity of IgG1 F12-MMAE, Ab1-mmae with comparison with the naked IgG1 F12 and Ab1 antibody and the linker-payload combination small molecule (vc-mmae). Compounds were incubated with PC-3-PSCA or PC-3 cells for 4-5 days. The cell viability was detected by the Promega CellTiter-Glo® luminescent cell viability assay.

    Article Snippet: The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated anti-human Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRP-conjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (Sigma-Aldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)-conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130-101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514).

    Techniques: Liquid Chromatography with Mass Spectroscopy, SDS Page, Enzyme-linked Immunosorbent Assay, Binding Assay, Conjugation Assay, In Vitro, Comparison, Incubation, Cell Viability Assay

    In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: mAbs

    Article Title: Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

    doi: 10.1080/19420862.2024.2387240

    Figure Lengend Snippet: In vivo therapeutic effects of ADC IgG1 F12-MMAE in prostate cancer PC-3-PSCA xenograft mouse model.(a) inhibition of tumor growth by F12-MMAE at the dose of 1 mg/kg and 3 mg/kg, 2XQ1W. The NSG mice were s.C. grafted with PC-3-PSCA cells ( n = 5). ADC was intraperitoneally administered. Ab1-mmae was used as the isotype control. Tumor sizes were monitored twice a week; (b) the mouse survival across different treatment groups in (A). Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. (c) The therapeutic efficacy of F12-MMAE when dosed at 6 mg/kg, 2XQ1W. After ADC washout, tumor relapsed. When tumors regrew to 140 mm³(38 days post-first injection), a single injection of 3 mg/kg of ADC was administered to evaluate the response of ADC to relapsed tumors. (d) The mouse survival across different treatment groups in (c). The two-way ANOVA followed by Tukey correction was used for statistical analysis of tumor size. The comparison of mouse survival curves was done by the log-rank test. ns: p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: The following antibodies were purchased from vendors: horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Sigma-Aldrich, Cat# A8592-1 MG); HRP-conjugated anti-human Fc antibody (Sigma-Aldrich, Cat# A0170-1 ML); HRP-conjugated anti-M13 PIII antibody (Sino Biological, Cat# NC1883789); HRP-conjugated anti-mouse IgG (Sigma-Aldrich, Cat# A0168-1 ML); PSCA antibody 7F5 (Santa Cruz Biotechnology, Cat#sc -80,654); Phycoerythrin (PE)-conjugated anti-Flag tag antibody (Miltenyi Biotec, Cat# 130-101-576); PE-conjugated anti-human Fab kappa light chain (LC) antibody (ThermoFisher, Cat# MH10514).

    Techniques: In Vivo, Inhibition, Control, Drug discovery, Injection, Comparison