An historical review of selected functions of exogenous Nerve Growth Factor: selective binding, endocytosis, and axonal transpor

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 An historical review of selected functions of exogenous Nerve Growth Factor: selective binding, endocytosis, and axonal transport

An historical review of selected functions of exogenous Nerve Growth Factor: selective binding,
endocytosis, and axonal transport
Kahl S.B.,* Burton L.E., ** Hill G.C.,*** and McKee, C.A.*
* PhD, Chief Scientific Officer, Manzanita Pharmaceuticals, Inc., Woodside, CA, US; ** PhD, consultant to Manzanita
Pharmaceuticals, Inc., LGB Consulting, San Mateo, CA, US; *** PhD, consultant to Manzanita Pharmaceuticals, Inc.;
Director, Radiopharm Development and Translation, SpectronRx, Indianapolis, IN, US; * MBA, Manzanita
Pharmaceuticals, Inc., Woodside, CA, US.
1 EXECUTIVE SUMMARY
Introduction. Nerve Growth Factor (NGF) was discovered by Rita Levi-Montalcini MD PhD in 1952 [1]
(Hamburger 1949 [2]; Cohen 1954 [3]; Levi-Montalcini 1976 [4]; Aloe 2004, 2012 [5, 6]). Nerve Growth Factor
has been considered as a therapeutic agent for multiple, mostly neurodegenerative conditions (Rocco 2018[7]). This review does not consider NGF as a potential, directly acting therapeutic agent. Rather, the scientific case is considered for NGF as a delivery facilitating moiety, to target the intraneuronal environment of peripheral nerves.
Historical review: methodology. This review considered the published literature as to exogenous
administration of NGF selective binding, internalization (endocytosis or phagocytosis), and axonal transport. This is not a systematic, but an historical narrative that identifies references from key papers as the main process. The review was conducted independently of the review paper, “Receptor binding, internalization, and retrograde transport of neurotrophic factors” (Neet and Campenot 2001 [8]).
One outcome of this review was noting that most of the basic research into the selective binding,
internalization, and axonal transport of NGF appears to have been completed by 2000. Since then, research has focused on various defects involving these processes in disease.
Product in development: the Nerve Growth Factor-fluorescent dye conjugate. This review examines the published literature of selected pharmacokinetics (PK) of Nerve Growth Factor (NGF), namely the emphasis on the mechanism and timing of binding to its receptors, whether the endocytosis of conjugates was possible, and the velocity of absorption, or retrograde axonal transport. Separately, we compared the published literature to results to date of our fluorescent dye-NGF conjugate, 800-rhNGF.
We are developing 800-rhNGF, a conjugate in which a known fluorescent dye in the 800 nanometer (nm) region is attached directly to amino acids located on the surface of recombinant human Nerve Growth Factor, NGF (800-rhNGF). Proprietary synthetic protocols leave both the dye free to fluoresce and NGF free to bind to its high and low affinity receptors, both of which are known. The first indication for 800-rhNGF is as a surgical guidance tool, a nerve imaging agent to be used in radical prostatectomies, in which localized, mostly early-stage prostate cancer is resected surgically.
After this review of the NGF literature, two separate comparisons evaluated whether the published NGF literature supported (i) the results of 800-rhNGF observed in non-GLP (GLP, Good Laboratory Practice) in rat studies to date (n=46) - yes; and (ii) the criteria for clinical workflow, as defined to us unanimously in detailed due diligence questions to urologic cancer surgeons – yes. Not reviewed are the characteristics of an 800 NIR dye which would also meet clinical criteria. Nonclinical results of 800-rhNGF will be published separately. This review focuses solely on what is known of the pharmacokinetics and science of NGF.
• Binding mechanism of NGF partly explains selectivity. It is definitive that (i) NGF binds with high affinity
for TrkA receptors; (ii) NGF also binds to the low affinity, “pan-neurotrophin” p75 receptor; (iii) binding to TrkA occurs within 3-6 min, to p75 within seconds (in PC12 cells, Godfrey and Shooter 1986; Senger and Campenot 1997 [9, 10]); (iv) TrkA and relatively more p75 receptors (percentage unknown) are expressed at the distal ends of nerves (Godfrey and Shooter 1986 [9]); and (v) TrkA is genetically encoded, and is highly homologous in all mammals, including humans ([11-13]).

For example, in an oncological resection of the prostate, the peri-prostastic space (“bed” of the initial surgical incision) enables access to TrkA and p75 receptors newly exposed after surgical incision. The other part of selectivity is that 800-rhNGF will ‘pool’ in the surgical space created by the incision. The relative benefits of localized vis-à-vis systemic administration are not reviewed here.
Table 1. Criteria for clinical utility for a nerve imaging agent to aid radical prostatectomies
Application At beginning of procedure, intra-operatively, interstitially (topically) to the peri-prostatic space (bed of initial surgical incision). No need to target any particular anatomical feature;
Wash Wash with saline three times (3X) after 15-30 min;
Durable Can be imaged at end of procedure, ~ 2h; Safe Degrades safely, so patient can be sutured up at the end of ~ 2h surgery; Clinical goal Does not interfere with primary goal, which is cancer control (does not tell surgeons what to do); and Benefits Intra-operative, intrastitial (topical, peri-prostatic) application should reduce systemic dose. Localized application of 800-rhNGF is likely to reduce patient in-surgery time, and reduce hospital cost if less time is spent in surgery and/or in hospital, if a day in hospital is spent if a nerve imaging agent is injected intravenously (IV). Not all nonclinical experiments distinguished carefully between whether the pro or mature form of NGF was used. It is critically important to understand this selection, since only the mature form binds to TrkA receptors (Luberg 2015; Fahnestock 2001; Ioannou and Fahnestock 2017; Shekari and Fahnestock 2019
[14-17]). For example, the 800-rhNGF nerve imaging agent under development uses only the mature form of NGF. As discussed further in Appendix D, “oncogene” describes the pro form of NGF, which is produced endogenously in adult mammals [18], but proNGF does not bind TrkA.
Endocytosis. The term endocytosis correctly describes endocytosis (“internalization”) of all proteins. What is reviewed here is the endocytosis of NGF-TrkA-(p75). It is known that the NGF-TrkA complex is moved intraneuronally, specifically on a ‘surface-bound’ path, on the outside of microtubules (Peters 1968, 1991 [19,20]; Rodriquez Echandia 1968 [21]; Burton P.R. 1984 [22]; Ure and Campenot 1997 [23]; Garvalov 2006 [24]).
Not reviewed are the specific mechanisms or the destination sites of transport of anterograde transport. What is not definitive is what primary and secondary signals are sent after NGF-receptor binding, and by what, to initiate endocytosis and then fast and slow retrograde axonal transport. Key findings from this review are:
(i) NGF is endocytosed with receptors TrkA and/or p75;
(ii) The NGF-receptor complex is probably transported in clathrin-coated vesicles (Howe 2001 [25]; Brown 2013 [26]);
(iii) NGF has been previously modified to involve even larger complexes than by comparison, the 800-rhNGF conjugate under development (mature form NGF kDA 26.3 is bound to fluorescent dye, MW 1015); and (iv) Once endocytosed, NGF-TrkA is loaded onto the retrograde axonal transport system with relatively high - 85% - efficiency (Ure and Campenot 1997 [23]).
Axonal transport. It is definitive that:
(i) Axonal transport includes NGF and other proteins;
(ii) There are fast (Brady 1984, 1993 [27, 28]; Brady 1985b [29]; Treanor 1995 [30]; Senger and Campenot 1997 [10]; Butowt and von Bartheld 2009 [31]) and slow components of axonal transport (Ure and Campenot 1997 [23]; Senger and Campenot 1997 [10]). The half-life of slow, retrogradely transported NGF in vitro is estimated at 6h (Ure and Campenot 1997 [23]);
(iii) Slow and fast axonal transport begins retrogradely (from the periphery to the neuronal cell body) (Hendry 1974a [32]; Brimijoin and Helland 1976 [33]; Allen 1982 [34]; Brady 1982 [35]; Stenoien and Brady 1999
[36]; Butowt and von Bartheld 2009 [31]);
(iv) In the neuronal cell body, the NGF-receptor complex is degraded into non-toxic components primarily by
proteolysis - by nucleases, proteases, esterases, glycosidases, lipases, phosphatases and sulfatases

(Avers 1982 [37]; Sheeler 1983 [38]; Parton and Dotti 1993 [39]; Hosang and Shooter 1986 [40];
Vissavajjhala 1992 [41]; Neet and Campenot 2001 [8]; Boutilier 2008 [42]; Frampton 2012 [43]); and
(v) Degraded, non-toxic NGF-receptor fragments are moved anterogradely (“orthograde” or anterograde
axonal transport), back to the periphery, channeling various products into various neuronal channels (Sec
3.3 Stenoien and Brady 1999 [36]; Butowt and von Bartheld 2009 [31]).
Future publications of nonclinical results of 800-rhNGF. Nonclinical results of 800-rhNGF will be published
in the future. Significantly, since the expression of Trk receptors is highly homologous in all mammals, the non-
GLP results to date in rat are clinically predictive. After NGF binds to TrkA and/or p75 receptors, absorption of
the NGF-receptor complex continues after wash, when the NGF-TrkA complex is loaded onto the retrograde
axonal transport system. ‘What the surgeon sees’ in the display of an imaging system is the 800 dye - not
indocyanine green (ICG) (Vahrmeijer 2013 [44]).
Ninety-nine per cent of the installed base of imaging systems are designed to detect a dye that fluoresces in
the 800 nm region: indocyanine green (ICG). The ability to visualize the 800 dye in 800-rhNGF has been
confirmed to date not only in all rat studies (total n=103), but also in two canine studies (n=2) that evaluated
two different marketed imaging systems. Both those imaging systems were designed to detect ICG, and
approved for marketing. This review does not discuss imaging systems. Future publication of nonclinical
results of 800-rhNGF will include:
(i) Dose range-finding studies (completed in rat, 1.0 mg/ml for Dye-Adduct-Ratio DAR2);
(ii) Signal-to-Background Ratio (SBR) calculations, from nerve-to-muscle (not nerve-in-adipose tissue)
measurements. As expected, SBR results reflect ‘steady state’ and the larger cargo size of the DAR2
variant: the SBR DAR2 was only ~ 25% higher than DAR1, even though number of molecules doubled
in the DAR2 variant from the DAR1 variant. In addition to the bioconjugation parameters, key
determinants were also the “brightness” of 800-rhNGF and sensitivity of the imaging systems;
(iii) Histology, using co-localization, TrkA was confirmed as a binding mechanism for 800-rhNGF. Neither
the binding of NGF to p75 nor the TrkA-p75 complex were assessed histologically; and
(iv) Canine studies, within the terms of the Non-Disclosure Agreements (NDAs) signed with three firms to
date. A fourth has expressed interest.
Contents of this Review
• In Sections 1 – 3 below, we review the selective binding, endocytosis, and axonal transport of NGF.
• Section 4 summarizes key findings from this review.