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Discovery and Basic Research

(T1130-06-32) Humanized scFv Antibody Specific to an Extracellular Epitope of CCK-BR and Non-Opioid Therapies for Pain Management

Tuesday, October 22, 2024
11:30 AM – 12:30 PM MT
Purpose: Small single-chain Fragment variable (scFv) antibodies were designed based on the upregulation of cholecystokinin B receptor (CCK-BR) mRNA in ganglia of chronic trigeminal orofacial pain models in mice compared to controls (Kunamneni et al. 2023). CCKBR is also a target known to be upregulated in chronic pain, anxiety, and depression. scFvs are composed of VH and VL portions of IgGs joined with a flexible linker. They are 30% smaller than monoclonal antibodies allowing brain/nervous tissue penetration demonstrated by Western blot. Targets validated through qPCR undergo further verification via immunostaining and protein immunoblotting.

Methods: Generation of the Humanized scFv Antibodies - For the antibody humanization process, we were utilized our humanization process through CDR grafting technology to design 3 VH x 3 VL humanized variants for recombinant production, purification, and affinity measurement (for top 10) by ELISA/Octet against human CCK-B peptide. Top humanized candidates were selected for in vitro and in vivo validations. Humanized CCKBR scFv Efficacy - Humanized CCKBR scFv (4 mg/kg, i.p.) is given to mice with FRICT-ION trigeminal nerve injury and the effect on mechanical hypersensitivity tested with von Frey filaments (Fig. 2). The humanscFv is given in week 2 after model induction.  Humanized CCKBR receptor scFv antibody testing in a nerve neuropathic pain model demonstrates the product is highly efficacious for reduction the hypersensitivity pain measure. The scFv is tested when given once in week 2-4 to assess mechanical hypersensitivity. The humanscFv is highly effective in reducing ongoing hypersensitivity after nerve injury. Full reversal to baseline may occur in week 3 after treatment (6 weeks after model induction). Testing in the subsequent weeks allows for efficacy testing for reducing anxiety- and depression-like behaviors comorbid with pain. Trigeminal Nerve Injury (FRICT-ION) model was tested with humanized scFv treatments (Montera and Westlund, 2020). Comparison to Commercial CCKBR Inhibitor, LY225910 - Mice with FRICT-ION were given daily injections of CCKBR inhibitor, LY225910 (s.c., 10 mg/kg). On Days 1-8, mice were tested prior to dosing. Efficacy tests on Days 6-8 progressed equivalently to a single dose of the lead CCKBR hscFv HC2-LC3 given on Day 1 (Fig. 2B). DRG neuron electrophysiology methods - Following ethically obtained consent, hDRG were obtained from recently deceased organ donors at UNM Hospital as per UNM IRB-approved protocols. Cultures of hDRG neurons were prepared as described previously (Valtcheva et al., 2016) and cultured for up to 11 days in vitro (DIV). Electrophysiology recordings took place 3-11 DIV. 53 cells were selected that were that were naïve or vehicle (media or water) controls. The Effect of HC2-LC3 hCCKBR-scFv (LEAD) on human DRG neuron excitability and the effect of hCCKBR variant scFv treatment on CCKBR expression in human DRG neurons were determined (Fig. 3). The in vitro characterization of humanized scFv in murine DRG sensory neurons was also performed as described earlier (Kunamneni et al. 2023).

Results: Based on the success of the murine parent scFv scFv77-2 (Kunamneni et al. 2023), nine humanized scFv (hscFv) variants were generated specific to CCK-B peptide using in silico complementary determining regions (CDR) grafting method (Fig.1A). Using the T20 humanness score of these humanized scFv variants, very low immunogenicity is expected in patients.Soluble expression and ELISA analysis showed that four humanized scFv variants had the highest binding and could be purified from bacterial cells in large quantities (Fig. 1B). Affinity measurement by ELISA indicates binding affinity in the low nanomolar range, in comparison to the murine parental. The HC2-LC3 had more than 25-fold improvement in affinity compared with parental mscFv77-2 (Fig. 1B). Octet measurements further revealed that the lead HC2-LC3 antibody had binding kinetics of  KD = 3.21 × 10–9 M (Fig. 1C). A single dose of 3 of 4 humanized CCKBR scFvs generated significantly and durably reduced chronic mechanical hypersensitivity with an effect persisting through at least 7 weeks in both male and female mice with neuropathic pain (Fig. 2). Our biologic tools are non-addictive, prevent/reduce the development of anxiety- and depression-like behaviors, and have no side effects with multi-dosing or testing over a 0.16 mg/kg to 40 mg/kg dose range. After i.v. injection, brain concentrations of the lead HC2-LC3 hscFv increase with time, providing peak concentration at 1 hr (2 µg/ml), a decline after Cmax and low-level presence through 24 hours. The plasma PK profile following subcutaneous and intravenous dosing persisted >24 hours. Whole-cell electrophysiology validation in vitro performed with human donor and mouse DRG neurons showed reduced excitability following treatment with 3 scFv variants (Fig. 3). RNA Sequencing revealed changes in several pain-related genes following scFv treatment of human DRG neurons.

Conclusion: Thus, the lead hscFv being tested has promising biotherapeutic applications for future treatment of chronic neuropathic pain.

References: Kunamneni, A.; Montera, M.A.; Durvasula, R.; Alles, S.R.A.; Goyal, S.; Westlund, K.N. Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain. Int. J. Mol. Sci. 2023, 24, 11035. https://doi.org/10.3390/ijms241311035
Montera MA, Westlund KN. Minimally Invasive Oral Surgery Induction of the FRICT-ION Chronic Neuropathic Pain Model. Bio Protoc. 2020 Apr 20;10(8):e3591. doi: 10.21769/BioProtoc.3591. PMID: 33659557; PMCID: PMC7842532.
Lyons DN, Zhang L, Pandya JD, et al. Combination Drug Therapy of Pioglitazone and D-cycloserine Attenuates Chronic Orofacial Neuropathic Pain and Anxiety by Improving Mitochondrial Function Following Trigeminal Nerve Injury. Clin J Pain. 2018;34(2):168-177. doi:10.1097/AJP.0000000000000515
Valtcheva M. V., Copits B. A., Davidson S., Sheahan T. D., Pullen M. Y., McCall J. G., et al. (2016). Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat. Protoc. 11 1877–1888. 10.1038/nprot.2016.111

Acknowledgements: The authors acknowledge the financial support of NIH grant R21 DE028096 (K.N.W., A.K.), the University of New Mexico Department of Anesthesiology & Critical Care Medicine endowment funds (K.N.W., S.A.), and NIH HEAL UG3 grant UG3 NS123958-01 (K.N.W., S.A., A.K.)

Fig. 1. (A) Antibody humanization using CDR grafting technology. (B) Human CCK-B peptide binding affinity of purified humanized and parental scFvs, HC1-LC2, HC2-LC1, HC2-LC3, HC3-LC3 and mscFv77-2. Dilutions of the purified antibodies were added to microtiter wells coated with human CCK-B peptide (1 µg/ml). Bound antibodies were detected by HRP-conjugated mouse anti-His tag antibody. Each point represents the mean ± s.d. values of triplicate wells. (C) Lead, HC2-LC3 and human CCK-B peptide affinity determination on the Octet RED384 instrument.


Fig 2. (A) Humanized CCKBR scFv Variants Relieve Mechanical Hypersensitivity. (B) Comparator Drug Treatment with CCKBR Inhibitor, LY225910.


Fig. 3. (A) Humanized CCKBR scFv variants reduce firing frequency of mouse DRG neurons. (B) Effect of HC2-LC3 hCCKBR-scFv (LEAD) on human DRG neuron excitability. (C) Effect of hCCKBR variant scFv treatment on CCKBR expression in human DRG neurons. Treatment with scFv was performed at a concentration of 10 ug/ml for 1 hr prior to fixation. 1:1000 CCKBR antibody from Alomone labs overnight at RT, and 1:1000 goat anti rabbit AF488 for 1 hour at RT. 1 coverslip each from donor HS-11. No. of cells HC2-LC1 =18, HC1-LC2=16, HC2-LC3=15, control=10.