Filamin A binding proteins and uses thereof
The present invention encompasses filamin A (FLNA) binding proteins. Specifically, the invention relates to antibodies to FLNA. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. Methods of making and methods of using the antibodies of the invention in methods of diagnosis, monitoring and prognosis of prostate cancer are also provided.
1. A method for detecting and/or quantifying the level of FLNA in a sample, comprising contacting the sample with an antibody construct, or antigen-binding fragment thereof, capable of binding filamin A (FLNA) comprising
(i) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10; or
(ii) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 16; or
(iii) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 22 under conditions such that the antibody construct or antigen-binding fragment thereof binds to FLNA in the sample, to thereby detect and/or quantify the level of FLNA in a sample.
2. The method of claim 1 , comprising detecting and/or quantifying one or more surrogate peptides in a protein digest prepared from said sample using mass spectrometry, wherein the mass spectrometry is multiple reaction monitoring (MRM) mass spectrometry.
3. The method of claim 2 , wherein the MRM is immunoprecipitation-multiple reaction monitoring (IPMRM) comprising a FLNA immunoprecipitation step.
4. The method of claim 2 , wherein the one or more surrogate peptides comprise the amino acid sequence of SEQ ID NO:40 (P2) and/or SEQ ID NO:41 (P4).
5. The method of claim 4 , wherein the range of detection for P2 is 62.5 pg/mL to 1500 pg/mL and the range of detection for P4 is 563 pg/mL to 27000 pg/mL.
6. The method of claim 4 , wherein a P2 internal standard (P2_IS) and/or a P4 internal standard (P4_IS) is also detected.
7. The method of claim 6 , wherein the MRM comprises monitoring one or more mass transitions m/z selected from the group consisting of: 441.7 (M+2H) 2+ →584.5 (y 5 1+ ) for P2; 535 (M+3H) 3+ →832.4 (y 8 1+ ) for P4, 445.5 (M+2H) 2+ →592.1 (y 5 1+ ) for the P2 internal standard (P2_IS), and 538.4 (M+3H) 3+ →842.5 (y 8 1+ ) for the P4 internal standard P4_IS.
8. A method for detecting and/or quantifying the level of FLNA in a sample, comprising detecting and/or quantifying one or more surrogate peptides, wherein the one or more surrogate peptides comprise the amino acid sequence of SEQ ID NO:40 (P2) and/or SEQ ID NO:41 (P4), in a protein digest prepared from said sample using mass spectrometry, wherein the mass spectrometry is multiple reaction monitoring (MRM) mass spectrometry, wherein the MRM is immunoprecipitation-multiple reaction monitoring (IPMRM) comprising a FLNA immunoprecipitation step, and wherein the immunoprecipitation step is carried out using an antibody construct, or antigen-binding fragment thereof, capable of binding filamin A (FLNA) comprising
(i) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10; or
(ii) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 16; or
(iii) a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 22.
9. The method of claim 8 , wherein the range of detection for P2 is 62.5 pg/mL to 1500 pg/mL and the range of detection for P4 is 563 pg/mL to 27000 pg/mL.
10. The method of claim 8 , wherein a P2 internal standard (P2_IS) and/or a P4 internal standard (P4_IS) is also detected.
11. The method of claim 10 , wherein the MRM comprises monitoring one or more mass transitions m/z selected from the group consisting of: 441.7 (M+2H) 2+ →584.5 (y 5 1+ ) for P2; 535 (M+3H) 3+ →832.4 (y 8 1+ ) for P4, 445.5 (M+2H) 2+ →592.1 (y 5 1+ ) for the P2 internal standard (P2_IS), and 538.4 (M+3H) 3+ →842.5 (y 8 1+ ) for the P4 internal standard P4_IS.