Safety and Feasibility of Cultivated Autologous Limbal Epithelial Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency (LSCD)
Phase: PHASE1
Conditions: Limbal Stem Cell Deficiency
Sponsor: Massachusetts Eye and Ear Infirmary

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 1 of 51 
 
SAFETY AND FEASIBILITY OF CULTIVATED AUTOLOGOUS LIMBAL EPITHELIAL CELL 
TRANSPLANTATION IN THE TREATMENT OF LIMBAL STEM CELL DEFICIENCY 
 
Protocol #: 14-124H 
FDA IND #: 16102 
Phase: I/II 
Version and Date:  
6.0 02/3/2022 
 
Principal Investigator and Enrolling Site: 
Ula Jurkunas, MD 
Massachusetts Eye and Ear Infirmary 
243 Charles Street 
Boston, MA 02114 
 
Co-Investigator: 
Reza Dana, MD, MPH, MSc 
Massachusetts Eye and Ear Infirmary 
243 Charles Street 
Boston, MA 02114 
 
Jia Yin, MD, PhD 
Massachusetts Eye and Ear Infirmary 
243 Charles Street 
Boston, MA 02114 
 
Collaborators: 
Jerome Ritz, MD 
Connell and O’Reilly Families 
Cell Manipulation Core Facility 
450 Brookline Ave  
Boston, MA 02215 
 
Allison Ayala, MS 
Jaeb Center for Health Research 
15310 Amberly Drive Ste 350 
Tampa, FL 33647 
 
Myriam Armant, PhD 
TransLab 
61 Binney Street 
208 Enders Building 
Boston, MA 02115 
 
 Funding Support: 
NIH/NEI through UG1 grants 
UG1EY026508 (Massachusetts Eye and Ear Infirmary) 
UG1EY027726 (Cell Manipulation Core Facility) 
UG1EY027725 (Coordinating Center) 
 
Past Funding Support: 
Production Assistance for Cellular Therapies (PACT) 
 of the National Heart, Lung, and Blood Institute  
National Institutes of Health 
Curing Kids Fund 
Massachusetts Lions Eye Research Foundation 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 2 of 51 
List of Abbreviations 
 
AE 
 
Adverse Event 
AIDS 
 
Acquired Immune Deficiency Syndrome 
AMT 
 
Amniotic Membrane Transplantation 
AS-OCT 
 
Anterior Segment Optical Coherence Tomography 
BCVA 
 
Best Corrected Visual Acuity 
BUN 
 
Blood, Urea, Nitrogen 
CALEC 
 
Cultivated Autologous Limbal Epithelial Cells 
CLAU 
 
Conjunctival Limbal Autograft 
CMCF 
 
Cell Manipulation Core Facility 
CRF 
 
Case Report Form 
eCRF 
 
Electronic Case Report Form 
DFCI 
 
Dana-Farber Cancer Institute 
eGFR  
 
Estimated Glomerular Filtration Rate 
FDA 
 
Food and Drug Administration 
GA 
 
General Anesthesia 
HIPAA 
 
Health Insurance Portability and Accountability Act 
HIV 
 
Human Immunodeficiency Virus 
ICF 
 
Informed Consent Form 
IND 
 
Investigational New Drug 
IRB 
 
Institutional Review Board 
IVCM 
 
In Vivo Confocal Microscopy 
LSC 
 
Limbal Stem Cells 
LSCD 
 
Limbal Stem Cell Deficiency 
MEEI 
 
Massachusetts Eye and Ear Infirmary 
MIVA 
 
Monitored Intravenous Anesthesia 
MM 
 
Medical Monitor 
MOP 
 
Manual of Procedures 
MRSA 
 
Methicillin-resistant Staphylococcus aureus 
NEI 
 
National Eye Institute 
OSDI 
 
Ocular Surface Disease Index 
PACT 
 
Production Assistance for Cellular Therapies 
PHRC 
 
Partners Human Research Committee 
QC 
 
Quality Control 
SANDE 
 
Symptom Assessment iN Dry Eye 
SOP 
 
Standard Operating Procedures 
 
 
 
 
 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 3 of 51 
 
Contents 
1. BACKGROUND ..................................................................................................................... 6 
2. STUDY AIMS ......................................................................................................................... 9 
2.1. Primary Objectives .................................................................................................. 9 
2.2. Secondary Objectives .............................................................................................. 9 
2.3. Study Conclusions ................................................................................................... 9 
3. STUDY TIMELINE ................................................................................................................10 
4. STUDY ORGANIZATION .....................................................................................................11 
5. POPULATION AND RECRUITMENT ...................................................................................12 
5.1. Participant Inclusion Criteria ...................................................................................12 
5.2. Participant Exclusion Criteria ..................................................................................12 
5.2.1. General Exclusion Criteria ...................................................................................12 
5.2.2. Exclusion Based on Either Eye ............................................................................13 
5.2.3. Exclusion Based on Donor Eye ...........................................................................13 
5.2.4. Exclusion Based on Recipient Eye ......................................................................13 
5.3. Recruitment ............................................................................................................13 
5.4. Informed Consent Process .....................................................................................13 
5.5. Enrollment Procedures ...........................................................................................14 
5.6. Second Biopsy........................................................................................................15 
5.7. Discontinuation of Study Follow Up And Participants Who Do Not Complete a 
Transplant .....................................................................................................................15 
5.8. Termination of Study...............................................................................................16 
6. STUDY PROCEDURES, MEASUREMENTS, AND VISIT SCHEDULE ................................17 
6.1. CALEC Manufacturing ............................................................................................17 
6.2. Preparation and Administration of Study Treatments ..............................................17 
6.2.1. Preparation (CALEC) ...........................................................................................17 
6.2.1.1. Limbal Biopsy ...................................................................................................17 
6.2.1.2. Transplantation .................................................................................................17 
6.2.2. Administration ......................................................................................................17 
6.2.2.1. CALEC Procedure – Biopsy ..............................................................................17 
6.2.2.2. CALEC Procedure – Transplantation ................................................................18 
6.3. Modification of Study Intervention for a Participant .................................................18 
6.4. Additional Surgeries ...............................................................................................19 
6.5. Medications and Treatments ...................................................................................19 
6.5.1. Concomitant Medication/Treatments ...................................................................19 
6.5.2. Acceptable Concomitant Medications ..................................................................19 
6.5.3. Prohibited Concomitant Medications ....................................................................19 
6.5.4. Study Medication Regimen ..................................................................................19 
6.5.5. Other Treatments ................................................................................................20 
6.6. Clinical Evaluations ................................................................................................20 
6.6.1. Symptom Assessment .........................................................................................21 
6.6.1.1. Ocular Surface Disease Index (OSDI) ..............................................................21 
6.6.1.2. Symptom Assessment iN Dry Eye (SANDE) .....................................................21 
6.6.2. Visual Acuity ........................................................................................................21 
6.6.3. Intraocular Pressure (IOP) Evaluation .................................................................21 
6.6.4. Slit Lamp Examination .........................................................................................21 
6.6.4.1. Corneal Opacification .......................................................................................22 
6.6.4.2. Corneal Fluorescein Staining ............................................................................22 
6.6.5. Schirmer’s Test ....................................................................................................22 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 4 of 51 
6.6.6. Impression Cytology ............................................................................................22 
6.6.7. Slit Lamp Photography ........................................................................................22 
6.6.7.1. Photography for Neovascular Area ...................................................................22 
6.6.7.2. Photography for Epithelial Defect Area .............................................................23 
6.6.8. Lid Margin and Conjunctiva Cultures ...................................................................23 
6.6.9. Fundus Examination ............................................................................................23 
6.7. Laboratory Evaluations ...........................................................................................24 
6.8. Study Schedule ......................................................................................................24 
6.8.1. Study Screening ..................................................................................................25 
6.8.2. Baseline (Within 30 days of Screening Visit) ........................................................26 
6.8.3. Limbal Biopsy (Within 25 Days of Baseline VISIT) ...............................................27 
6.8.4. Transportation of Tissue to CMCF & Preparation of CALEC ................................27 
6.8.5. Post-Biopsy Assessment of Donor Eye (1-2 Days after CALEC Biopsy) ..............27 
6.8.6. Transportation of CALEC from CMCF to MEEI ....................................................27 
6.8.7. Preoperative Assessment of Recipient Eye (1 to 5 Days Prior to Transplant) ......27 
6.8.8. Corneal Reconstruction with CALEC (Day 0) .......................................................27 
6.8.9. Follow-up Period ..................................................................................................28 
6.8.10. Post-Transplant (Day 1) .....................................................................................28 
6.8.11. Post-Op Follow-up: Weeks 1, 2, Month 1 (4 WEEKS ± 3 days) and Months 3, 6, 
9, 12 (13, 26, 39, 52 WEEKS ± 1 week), and Months 15, and 18 (65 and 78 WEEKS ±2 
weeks) ...........................................................................................................................28 
6.8.12. Early Termination Visit .......................................................................................29 
6.8.13. Unscheduled Visit ..............................................................................................29 
7. OUTCOME MEASURES .......................................................................................................30 
7.1. Primary Outcome Measures ...................................................................................30 
7.1.1. Safety Measures ..................................................................................................30 
7.1.2. Feasibility Measures ............................................................................................30 
7.2. Secondary Outcome Measures ..............................................................................30 
7.3. Other Outcome Measures ......................................................................................31 
8. MONITORING STUDY PROGRESS .....................................................................................32 
9. ASSESSMENT OF SAFETY ................................................................................................33 
9.1. Methods and Timing for Assessing, Recording, and Reporting of Safety Parameters
 ......................................................................................................................................33 
9.2. Safety Oversight .....................................................................................................33 
9.3. Study Halting Guidelines ........................................................................................33 
10. MONITORING PROTOCOL ADHERENCE ........................................................................35 
11. QUALITY CONTROL OF DATA .........................................................................................36 
12. CONTINUED FOLLOW UP FOR CLAU PARTICIPANT ENROLLED PRIOR TO 
PROTOCOL AMENDMENT DISCONTINUING CLAU ARM OBJECTIVE ................................37 
13. LITERATURE REFERENCES ............................................................................................38 
14. APPENDICES .....................................................................................................................41 
14.1. APPENDIX I: Symptom Measurement Scales ......................................................41 
14.2. APPENDIX II: National Eye Institute Corneal Fluorescein Grading Scale .............44 
14.3. APPENDIX III: Vascularization Measurement Tool ...............................................45 
14.4. APPENDIX IV: Fantes Scale for Corneal Opacity .................................................46 
14.5. APPENDIX V: Impression Cytology ......................................................................47 
14.6. APPENDIX VI: Schedules of Events & Procedures For Participants Completing 
Transplant .....................................................................................................................48 
15. PROTOCOL ADDENDUM: SECOND TRANSPLANT ........................................................49 
15.1. OVERVIEW ..........................................................................................................49 
15.2. CRITERIA FOR SECOND TRANSPLANT ............................................................49 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 5 of 51 
15.3. INFORMED CONSENT PROCESS ......................................................................49 
15.4. SCREENING AND BASELINE .............................................................................49 
15.5. BIOPSY AND TRANSPLANT ...............................................................................50 
15.6. STUDY PROCEDURES, MEASUREMENTS, AND VISIT SCHEDULE ................51 
15.7. ANALYSIS CONSIDERATIONS ...........................................................................51 
15.7.1. SAFETY ............................................................................................................51 
15.7.2. FEASIBILITY .....................................................................................................51 
15.7.3. EFFICACY .........................................................................................................51 
 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 6 of 51 
1. BACKGROUND 
1 
The cornea is a transparent, avascular tissue covered by non-keratinized stratified epithelium 
2 
that is responsible for maintaining a smooth ocular surface for normal vision as well as for 
3 
providing a barrier against environmental and external stress. The entire ocular surface is 
4 
covered by corneal, limbal, and conjunctival epithelial cells that, together with a stable pre-
5 
ocular tear film, maintain its integrity.   
6 
 
7 
Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent 
8 
the ultimate source of transparent corneal epithelium (Schermer et al., 1986, Tseng et al., 
9 
1996). When these limbal stem cells (LSC) become dysfunctional or deficient, the cornea is 
10 
unable to maintain its surface epithelial integrity and phenotype and a disease called corneal 
11 
limbal stem cell deficiency (LSCD) develops.  
12 
 
13 
Corneal scarring and opacity is the 5th commonest cause of blindness worldwide, accounting for 
14 
5.1% of blindness (Resnikoff et al., 2008). LSCD, a major cause of corneal scarring, arises from 
15 
a variety of congenital or acquired causes that are infectious (like trachoma), immunologic, 
16 
oncologic or iatrogenic in nature and in turn lead to severe ocular surface dysfunction. The 
17 
LSCD can be caused by Stevens-Johnson syndrome, ocular cicatricial pemphigoid, aniridia, 
18 
chemical or thermal burns, contact lenses, a variety of microbial infections, long-term use of 
19 
topical (including antiglaucoma) medications, irradiation, tumors, or multiple surgical procedures 
20 
or cryotherapy involving the ocular surface. LSCD afflicts thousands of people in North America 
21 
(Holland and Schwartz, 1999) and is particularly prevalent in chemical and thermal burns of the 
22 
ocular surface.  
23 
 
24 
The clinical hallmark of LSCD is conjunctivalization of the corneal surface (or replacement of 
25 
normal and transparent corneal epithelium by opaque conjunctival epithelium), 
26 
neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and 
27 
scarring all of which can lead to decreased vision, pain, and impaired quality of life 
28 
(Puangsricharern and Tseng, 1995) (Kruse et al., 1990) (Figure 1). 
29 
 
30 
 
 
 
 
31 
Figure 1 A and B: In limbal stem cell deficiency (LSCD), the cornea becomes vascularized, 
32 
scarred, and opaque; the demarcation between cornea and conjunctiva is lost. Patients 
33 
experience loss of vision, photophobia, irritation and pain. 
34 
Conventional corneal transplantation replaces only central cornea and cannot satisfactorily treat 
35 
the corneal surface with extensive or complete LSCD. As a result, therapeutic strategies have 
36 
been developed at replacing limbal epithelium with or without corneal transplantation surgery 
37 
(Tseng, 1996, Kenyon and Tseng, 1989, Copeland and Char, 1990, Morgan and Murray, 1996, 
38 
Holland and Schwartz, 1996). Several techniques have been reported for limbal stem cell 
39 
transplantation or keratoepithelioplasty. In unilateral cases of LSCD, donor tissue is obtained 
40 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 7 of 51 
from the fellow eye, called limbal autograft; in bilateral cases of LSCD  tissue is obtained from 
41 
cadaver donors, called limbal allograft (Yalcindag et al., 2008, Dua and Azuara-Blanco, 1999, 
42 
Kenyon and Tseng, 1989). Both procedures seek to provide therapeutic benefit through 
43 
transplantation of a new source of epithelium onto a diseased ocular surface after the removal 
44 
of the recipient’s scarred and diseased epithelium (Dua and Forrester, 1990, Dua and Azuara-
45 
Blanco, 1999, Clinch et al., 1992, Jenkins et al., 1993, Kenyon and Tseng, 1989). Since limbus 
46 
is vascularized and contains antigen presenting cells, limbal allografts carry a high risk of 
47 
rejection and require use of lifelong systemic immunosuppression. Limbal allograft survival is 
48 
limited to 50% at 5 years despite the use of systemic immunosuppression and survival is much 
49 
lower if concurrent penetrating keratoplasty is performed to aid in visual rehabilitation (Smolin, 
50 
2005). 
51 
 
52 
Autologous limbal transplantation, such as conjunctival limbal autograft (CLAU), is a viable 
53 
option in unilateral cases and it circumvents the use of immunosuppression. Traditionally, two 
54 
large pieces of the limbus are removed from the donor eye. The main limitation of this technique 
55 
is that it carries risks of inducing limbal stem cell deficiency to the donor eye when harvesting 
56 
limbal autografts (Haamann et al., 1998, Tan et al., 1996, Kenyon and Tseng, 1989). Cases of 
57 
corneal haze, pseudopterygium, epithelial defects and conjunctivalization in the donor eye have 
58 
been reported (Dua et al., 2010, Morgan and Murray, 1996, Fogla and Padmanabhan, 2005, 
59 
Yao et al., 2002). Although recently some attempts have been made to use a smaller graft, such 
60 
as Simple Limbal Epithelial Transplantation (SLET), there are only limited data, especially 
61 
regarding long-term follow-up on the new procedure (Sangwan, 2012). 
62 
 
63 
In order to circumvent risks of allograft rejection (as seen in limbal allografts) and damage to the 
64 
donor eye (as seen in limbal autografts, such as CLAU), a technique has been developed to 
65 
cultivate autologous limbal epithelial cells (CALEC) for transplantation. This technique is 
66 
advantageous because of its ability to utilize a small amount of the participant’s own tissue. The 
67 
risk of damage to the donor eye is bypassed by expanding limbal epithelial (stem) cells from a 
68 
small biopsy (2-3 mm2) onto the substrate, such as amniotic membrane, in culture before 
69 
transplantation. The success of using such a new surgical approach has been reported in 
70 
several human studies (Koizumi et al., 2001, Rama et al., 2010, Sangwan et al., 2005, Schwab 
71 
et al., 2000, Shimazaki et al., 2007, Shortt et al., 2007, Basu et al., 2012, Baradaran-Rafii et al., 
72 
2010, Nakamura et al., 2006, Pellegrini et al., 1997).  
73 
 
74 
The general advantages of CALEC transplantation are that 1) it provides an amplified source of 
75 
epithelial (stem) cells as compared to transplantation of the similar size of limbal biopsy, 2) it is 
76 
an autologous source of cells and does not carry risk of immunologic rejection, 3) it carries 
77 
significantly lower risk of inducing LSCD in the donor eye, and 4) it is possible to repeat the 
78 
biopsy and transplant owing to the non-invasiveness of the procedures to both donor and 
79 
recipient eyes. Currently there are no good options for treatment of LSCD in the U.S and 
80 
patients have a significant burden from the disease. That, along with recent advances in stem 
81 
cell research provide compelling  reasons for conducting a study aimed at assessing first, the 
82 
safety and feasibility of CALEC grafts, and  additionally, their effectiveness in treating LSCD. 
83 
 
84 
The most common substrate utilized for ex vivo expansion of limbal epithelial cells has been 
85 
amniotic membrane. Amniotic membrane transplantation (AMT) has extensive history in ocular 
86 
surface reconstructive surgery and its utility alone or in combination with limbal stem cell 
87 
transplantation has been reported (Holland and Schwartz, 2000, Koizumi et al., 2000, Kolli et 
88 
al., 2010, Nakamura et al., 2003, Prabhasawat et al., 2001, Shimazaki et al., 2002). In cases of 
89 
partial LSCD, AMT alone has been shown to provide a useful substrate for regeneration of the 
90 
remaining epithelial cells by enhancing epithelial cell migration and creating a hospitable 
91 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 8 of 51 
microenvironment mimicking limbal stem cell niche (Grueterich et al., 2003, Tseng, 1989, 
92 
Dietrich-Ntoukas et al., 2012, Shahdadfar et al., 2012, Tsai and Tsai, 2010, Tseng et al., 2002). 
93 
In addition, use of amniotic membrane as a substrate for limbal epithelial cell growth has added 
94 
the benefits of amnion’s ability to facilitate epithelialization (Lee and Tseng, 1997, Tsai and 
95 
Tseng, 1988), reduce inflammation and scarring (Fernandes et al., 2005, Prabhasawat et al., 
96 
2001), and act as a new and natural basement membrane when the underlying stromal tissue 
97 
has been destroyed. 
98 
 
99 
Combining the expansion of cells ex vivo with cultivation on an amniotic membrane has the 
100 
advantage of ensuring a compatible extracellular matrix for the graft, thus increasing its 
101 
durability and manipulability (Zakaria et al., 2010). This method is an improvement over earlier 
102 
attempts at the use of engineered corneal surfaces, in which fragile sheets of epithelial cells, 
103 
with no substantial underlying stromal support, were transplanted (Nishida et al., 2004, 
104 
Pellegrini et al., 1997). 
105 
 
106 
There is no CALEC product available in the U.S., and we have thoroughly reviewed efforts to 
107 
produce such a product in other countries. As a result, our first goal has been to optimize and 
108 
standardize the techniques of CALEC preparation for the clinical trial in the U.S. We have 
109 
significantly improved upon the quality of the cell therapy product and process of preparation by 
110 
using completely defined reagents, preforming additional testing on reagents used (for example 
111 
bovine adventitious virus testing), using autologous starting material, removing antibiotics and 
112 
murine feeder cells, employing highly reproducible methods for cell isolation, and expansion and 
113 
quality control of the resultant CALEC sheets. These modifications and standardizations were 
114 
aimed at enhancing safety and predictability of the patients’ clinical outcomes. Based on the 
115 
pre-clinical testing performed in the Center for Human Cell Therapy Laboratory the CALEC 
116 
constructs are ready for the phase I/II clinical trial. We have received Investigational New Drug 
117 
Application (IND #16102) approval from the FDA for our CALEC graft to perform the clinical trial 
118 
at the Massachusetts Eye and Ear Infirmary (MEEI) for unilateral LSCD. The FDA has advised 
119 
us in the setting of aims, inclusion and exclusion criteria, and study design. 
120 
 
121 
In summary, LSCD is a common cause of serious and prevalent corneal blindness, and there 
122 
are significant limitations to the current available treatment options and standards of care.  
123 
Taking into consideration prior experience with CALEC in other countries, we have set a goal to 
124 
optimize and standardize the techniques of CALEC preparation for the clinical trial in the U.S. 
125 
We have significantly improved upon the quality of the cell therapy product and process of 
126 
preparation; such modifications and standardizations are aimed at maximizing safety and 
127 
predictability of the participants’ clinical outcomes. In this application, we will evaluate the safety 
128 
and feasibility of our CALEC product as well as its efficacy in treating the LSCD. 
129 
 
130 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
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2. STUDY AIMS 
131 
The main aim of the study is to determine preliminary estimates of the safety and feasibility of 
132 
cultivated autologous limbal epithelial cell (CALEC) transplantation in the treatment of unilateral 
133 
LSCD. Secondarily, efficacy of CALEC will be investigated. This secondary objective is intended 
134 
to be exploratory rather than conclusive.  
135 
 
136 
2.1. PRIMARY OBJECTIVES 
137 
1. Safety: To establish the safety of CALEC transplantation by determining the incidence of 
138 
primary ocular adverse events through 18 months of follow-up.  
139 
 
140 
2. Feasibility: To establish feasibility of manufacturing CALEC for corneal surface 
141 
reconstruction. 
142 
 
143 
2.2. SECONDARY OBJECTIVES 
144 
 
145 
1. Efficacy: To investigate whether CALEC transplantation is efficacious in treatment of LSCD 
146 
participants by comparing pre-operative to post-operative clinical parameters at months 3, 
147 
12, and 18. 
148 
 
149 
 
150 
2.3. STUDY CONCLUSIONS 
151 
The results of this Phase I/II study will provide guidance on whether to continue to a larger 
152 
Phase III study and will also provide preliminary data to help determine sample size for future 
153 
trials. Generally, if the study is not halted for feasibility issues, and there are no major safety or 
154 
efficacy concerns, a larger study of CALEC could be considered. However, caveats of the study 
155 
data and other potential factors outside of the trial will need to be considered and weighed in the 
156 
ultimate decision of whether to proceed.   
157 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 10 of 51 
3. STUDY TIMELINE 
158 
The total duration of the study, from funding start date to final analysis and manuscript, will be 
159 
approximately 72 months. This will include study startup, initiation and completion of recruitment 
160 
(according to procedures detailed in Section 5), completion of 18 months of follow-up for all 
161 
participants completing a transplant, and study closeout. Details of the study timeline and 
162 
milestones can be found in the CALEC Study Policy Document. 
163 
 
164 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 11 of 51 
4. STUDY ORGANIZATION 
165 
This is an open label, single-center study to assess safety and feasibility of CALEC 
166 
transplantation in participants with unilateral LSCD. This study will be performed at the Clinic 
167 
Center, Massachusetts Eye and Ear Infirmary in Boston, MA. The CALEC will be manufactured 
168 
in the Connell and O’Reilly Families Cell Manipulation Core Facility (CMCF) at the Dana-Farber 
169 
Cancer Institute (DFCI) in Boston, MA, which will be the Resource Center for this study (CMCF 
170 
DFCI). The Coordinating Center for this study will be Jaeb Center for Health Research located 
171 
in Tampa, FL. 
172 
  
173 
We will also have two Committees for this study, the Operations Committee and the Data and 
174 
Safety Monitoring Committee (DSMC). The Operations Committee, which also functions as both 
175 
Executive and Editorial Committees, has the overall responsibility for administering the study 
176 
and for making day-to-day operational decisions including the recruitment plan, data capture 
177 
and maintenance, protocol compliance, participant retention, and planning of investigator and 
178 
other committee meetings. The DSMC will be responsible for reviewing the ethical conduct of 
179 
the study and for monitoring the data for evidence of adverse or beneficial treatment effects. 
180 
Details of the study organization can be found in the CALEC Study Policy Document. 
181 
 
182 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
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5. POPULATION AND RECRUITMENT 
183 
This study includes participants with unilateral LSCD. All participants will be recruited at the 
184 
Massachusetts Eye and Ear Infirmary according to Section 5.3. All study participants will be 
185 
screened and must fit all criteria described in Sections 5.1 and 5.2. Should a participant’s first 
186 
biopsy fail, the participant can be rescreened at a later date for possible participation (Section 
187 
5.6).  
188 
 
189 
5.1. PARTICIPANT INCLUSION CRITERIA   
190 
• 
Male or female participants age 18 to <90 years old at time of enrollment with a minimum life 
191 
expectancy of 18 months 
192 
• 
Ability of a participant or guardian/legal representative to provide written informed consent 
193 
and to comply with study assessments for the full duration of the study 
194 
• 
Participants with unilateral limbal stem cell deficiency (LSCD) as determined by 
195 
conjunctivalization of the cornea defined by fibrovascular pannus more than 2 mm from the 
196 
limbus into the cornea for ≥6 clock hours 
197 
o Ideal candidates will also meet the following additional criteria, but these will not be 
198 
required:  
199 
o Lack of limbal palisades of Vogt for ≥9 clock hours 
200 
 
201 
5.2. PARTICIPANT EXCLUSION CRITERIA  
202 
5.2.1. GENERAL EXCLUSION CRITERIA  
203 
Confirmed none of the following are present via blood draw at screening visit: 
204 
• 
Uncontrolled diabetes, defined as most recent HbA1c >8.5% (does not need to be repeated 
205 
at screening visit if done within the last 3 months prior to screening visit) 
206 
• 
Decreased renal function, defined as eGFR below 60 mL/min per 1.73 m2 
207 
• 
Aspartate aminotransferase or alanine aminotransferase levels >3x institutional upper limit 
208 
of normal (Institutional upper limit: AST 10-40 U/L ALT 10-55 U/L) 
209 
• 
Total bilirubin >2.0x institutional upper limit of normal (except participants with known 
210 
Gilbert’s syndrome) (Institutional upper limit: Total Bilirubin 0.0-1.0 mg/dL) 
211 
• 
Platelet levels <100,000 or >450,000 per microliter 
212 
• 
Hemoglobin levels <11.0 g/dL in men <10.0 g/dL in women 
213 
• 
Prothrombin time >16 seconds or activated partial thromboplastin time >35 seconds in 
214 
participants not taking warfarin or an international normalized ratio >3 in participants taking 
215 
warfarin 
216 
• 
Human Immunodeficiency Virus (HIV) infection or Acquired Immune Deficiency Syndrome 
217 
(AIDS) 
218 
• 
Active hepatitis B or C 
219 
 
220 
Other criteria: 
221 
• 
Inability to tolerate monitored anesthesia 
222 
• 
Current pregnancy (positive urine test) or lactation, or intent to become pregnant between 
223 
enrollment and the first 3 months after transplant 
224 
• 
Participation in another simultaneous medical investigation or trial 
225 
• 
Any medical, psychiatric, debilitating disease/disorder or social condition that in the 
226 
judgment of the investigator would interfere with or serve as a contraindication to adherence 
227 
to the study protocol or ability to give informed consent 
228 
• 
Signs of current infection, including fever and current treatment with antibiotics 
229 
• 
Presence of potential allergy to the CALEC graft or any of the chemical components within 
230 
its formulation 
231 
 
232 

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5.2.2. EXCLUSION BASED ON EITHER EYE 
233 
• 
Prior corneal surgery within 30 days prior to study entry except placement of amniotic 
234 
membrane 
235 
• 
Corneal or ocular surface infection within 30 days prior to study entry or CALEC type 
236 
transplantation 
237 
• 
Ocular surface malignancy 
238 
• 
Severe cicatricial eye disease 
239 
 
240 
5.2.3. EXCLUSION BASED ON DONOR EYE 
241 
• 
Conjunctivalization of the cornea defined by fibrovascular pannus more than 2mm from the 
242 
limbus into the cornea for ≥3 clock hours 
243 
• 
Lack of limbal palisades of Vogt for ≥3 clock hours 
244 
• 
History of allo-limbal transplantation 
245 
• 
Severe dry eye disease as determined by Schirmer’s test ≤5 mm 
246 
 
247 
 
248 
5.2.4. EXCLUSION BASED ON RECIPIENT EYE 
249 
• 
Severe dry eye disease as determined by Schirmer’s test ≤2 mm 
250 
 
251 
5.3. RECRUITMENT 
252 
This study will be conducted in accordance to the Food and Drug Administration’s (FDA) 
253 
regulations. In addition, this study will follow the guidelines set forth in the Health Insurance 
254 
Portability and Accountability Act (HIPAA), which are written in 45 CFR 160-164. 
255 
 
256 
Potential study participants will be identified by the study investigators and their research team 
257 
at the Cornea and Refractive Surgery Service, Massachusetts Eye and Ear Infirmary. A 
258 
Research Technician, Study Coordinator, Research Fellow and/ or Investigator will identify the 
259 
patients in their clinic who have unilateral LCSD. Once identified, the study team will evaluate 
260 
whether the patient may be able to participate in the study based on their medical record and 
261 
the study eligibility criteria. If no definitive reasons to exclude the patient are found during the 
262 
review, the patient will be approached by their physician (investigator) to review the study.   
263 
 
264 
Those patients who volunteer to participate will go over the study details and the consent form 
265 
with study staff.  If he or she understands the study, including its risks, and agrees to participate, 
266 
they will be asked to sign a written consent document. Subsequently, the screening procedures 
267 
will be performed to confirm the eligibility of the study participant. Once it is determined that the 
268 
participant qualifies to enroll in the study, he or she will have another visit to provide the 
269 
baseline data. After this baseline visit, the participant will be assigned into a study arm, as 
270 
described below in Section 5.5. The schedule of post-corneal reconstruction visits is standard 
271 
for participants undergoing these types of procedures.  
272 
 
273 
This study does not exclude, or intentionally recruit, participants based on gender, race, or 
274 
ethnicity. 
275 
 
276 
5.4. INFORMED CONSENT PROCESS 
277 
Participants are required to sign an informed consent form (ICF) which includes a Health 
278 
Insurance Portability and Accountability Act (HIPAA) authorization form before participating in 
279 
the study. Given that the Investigators and study staff are responsible for maintaining detailed 
280 
knowledge of the study protocol, safety profile, and previous work with the procedure, these 
281 
individuals will discuss the protocol with potential participants and obtain informed consent. The 
282 

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study team will review the study procedures, visit schedule, known risks, potential benefits (if 
283 
any), alternative treatments and financial responsibilities with all potential participants. Each 
284 
participant will be informed of their right to withdraw at any time from the study without affecting 
285 
their care or relationship with the treating physician and participating institution. A study member 
286 
will also explain and discuss with the participant their confidentiality rights as described in the 
287 
HIPAA form. Participants will be given the opportunities to ask questions. An Investigator will 
288 
obtain written consent from each participant prior to any study procedures performed by the 
289 
study team.  
290 
 
291 
A note will be made on the study record that the ICF was signed by the participant. The ICF will 
292 
follow the guidelines set forth by the FDA and Partners Human Research Committee (PHRC), 
293 
which is the Institutional Review Board (IRB) for this study. A copy of the signed consent form 
294 
will be given to the participant. 
295 
 
296 
We do not anticipate a need to obtain on-going consent over the course of the study. If protocol 
297 
amendments result in consent form changes, participants will be re-consented if the changes 
298 
affect data integrity, or affect the participants’ rights, safety, or well-being. Legally Authorized 
299 
Representatives (LAR) can consent on behalf of participants who cannot provide their own 
300 
consent. Consent by the LAR will be done in accordance with Massachusetts laws and PHRC 
301 
requirements. 
302 
 
303 
5.5. ENROLLMENT PROCEDURES 
304 
There will be two sequential phases of enrollment – Phase A followed by Phase B.  
305 
 
306 
Phase A – Staggered Enrollment 
307 
During Phase A, eligible participants completing the screening and baseline visits will be 
308 
enrolled in a staggered fashion (described below) to receive CALEC. This will be the procedure 
309 
until three participants in Phase A complete a CALEC transplant (or until halting guidelines 
310 
apply, see Section 9.4). During Phase A, the following will apply: 
311 
• 
If a participant enrolls during Phase A but withdraws from the study prior to transplant, the 
312 
participant will not count towards the recruitment goal of three participants required for 
313 
Phase A  
314 
• 
If a biopsy is done for a participant in Phase A, and the biopsy is unable to generate a viable 
315 
cell sheet for transplant, a second biopsy may be attempted according to the procedure in 
316 
Section 5.6. 
317 
• 
Once a given Phase A biopsy attempt has either (1) failed or (2) succeeded and participant 
318 
has completed the 2-week visit, the study team can then proceed to either of the following: 
319 
 Schedule a second biopsy attempt for any prior Phase A participant whose first 
320 
biopsy failed (according to Section 5.6) 
321 
 Consent and enroll a new participant in Phase A 
322 
 
323 
• 
Phase A enrollment ends after three participants in Phase A have completed a CALEC 
324 
transplant and the 2-week visit after transplantation.   After this occurs, Phase B will 
325 
begin.   Some additional Phase A participants whose first biopsy failed may complete a 
326 
second biopsy after Phase B begins, and would count towards the Phase A cohort.    
327 
 
328 
 
329 
Phase B – Open Enrollment 
330 
During Phase B, eligible participants completing the screening and baseline visits will be 
331 
enrolled to receive CALEC, with no requirements to stagger the enrollments.     
332 

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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
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333 
During Phase B, the following will apply:  
334 
• 
If a participant enrolls during Phase B but withdraws from the study prior to transplant, the 
335 
participant will not count towards the recruitment goal. 
336 
• 
If a biopsy is done for a participant in Phase B, and the biopsy is unable to generate a viable 
337 
cell sheet for transplant, a second biopsy can be attempted according to the procedure in 
338 
Section 5.6. 
339 
 
340 
 
341 
Study enrollment ends as soon as one of the following is met (whichever occurs first): 
342 
• 
A total of 17 CALEC transplants (across Phase A and B) have been completed. 
343 
• 
The last transplant that can reasonably allow 18 months of follow up within the study 
344 
timeline as noted in Section 3 has been completed.  
345 
 
346 
 
347 
The Statistical Analysis Plan documents how participants in the study cohort will be analyzed. 
348 
 
349 
In both Phases, investigators, participants, and those involved in the study assessments will all 
350 
be aware of (unmasked to) the study intervention received and appropriate protocol procedures. 
351 
 
352 
5.6. SECOND BIOPSY  
353 
If a participant experiences failure of the biopsy to generate a viable cell sheet, the reasons for 
354 
failure will be reviewed. The participant can be rescreened for a second attempt at biopsy and 
355 
CALEC construct if the following are met: (1) the reason for failure is not likely to re-occur for 
356 
that participant, (2) a repeat biopsy does not present a significant risk to the participant, and (3) 
357 
study enrollment has not ended.  Failure is considered not likely to re-occur when initial failure is 
358 
deemed secondary to culture condition not cell growth (such as evidenced by normal growth of 
359 
P0 cells). Second biopsy attempts will be scheduled at least 30 days after the initial biopsy 
360 
which is medically appropriate for this type of biopsy procedure. The screening visit and 
361 
baseline visit will be repeated, unless the second biopsy is scheduled to occur within 55 days of 
362 
the original screening visit date, in which case only the baseline visit will be repeated. The 
363 
second biopsy must occur within 25 days of the second baseline visit. (Note: The second 
364 
baseline assessments will not be used as primary baseline values,  but rather will be used to 
365 
document any changes, and may be considered for sensitivity analyses.) 
366 
 
367 
Each participant will be limited to a total of two biopsies. If a second attempt will not be pursued 
368 
or the second biopsy also fails to generate a viable cell sheet, the participant may or may not 
369 
continue in follow up, as defined by the criteria in Section 5.7. 
370 
 
371 
5.7. DISCONTINUATION OF STUDY FOLLOW UP AND PARTICIPANTS WHO DO NOT 
372 
COMPLETE A TRANSPLANT 
373 
A study participant has the right to withdraw from the study at any time.  If a study participant is 
374 
considering withdrawal from the study, the principal investigator should personally speak to the 
375 
individual about the reasons, and every effort should be made to accommodate him or her. 
376 
Participants that decide to withdraw after transplantation will be asked to return to the clinic for 
377 
an Early Termination Visit as described in Section 6.8.12. After discontinuation of study follow 
378 
up for any reason, the subject will continue care with their non-study clinician.     
379 
 
380 
Other considerations for study discontinuation are as follows: 
381 
• 
Prior to biopsy 
382 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
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o Study participants who are determined to be ineligible for any reason (including 
383 
pregnancy) prior to biopsy will not proceed to biopsy or transplant, and will either  
384 
discontinue or postpone and re-screen at a later time. 
385 
• 
After biopsy but prior to transplant  
386 
o Study participants who become pregnant will consult with the study doctor and their 
387 
Obstetrician/Gynecologist or primary care physician before determining if it is safe for 
388 
them to continue with the transplant procedure.  If the decision is made not to 
389 
continue with the transplant procedure, the participant will either discontinue or 
390 
postpone and re-screen at a later time. 
391 
o Study participants who are determined to be ineligible for any other reason will 
392 
consult with the study doctor before determining if it is safe for them to continue with 
393 
the transplant procedure.  If the decision is made not to continue with the transplant 
394 
procedure, the participant will either discontinue or postpone and re-screen at a later 
395 
time. 
396 
o Study participants for whom the biopsy fails to generate a viable cell sheet may be 
397 
evaluated for a second biopsy according to Section 5.6 above. If the participant does 
398 
not proceed to second biopsy for any reason, or a second biopsy also fails to 
399 
generate a viable cell sheet, the participant will discontinue. 
400 
o Additionally: Regardless of whether a participant will discontinue,  all participants 
401 
who had a biopsy and do not proceed to transplant will be monitored by the study 
402 
team for 30 days to check for any adverse events related to the biopsy procedures.   
403 
If the donor eye after biopsy has a complication or adverse event that does not 
404 
resolve after 30 days, the study team will also monitor the donor eye until the 
405 
complication resolves.  Those meeting criteria to discontinue, as detailed in the 
406 
scenarios above, will discontinue after this time.  
407 
• 
After transplant  
408 
o In general, all possible efforts will be employed to ensure participants who completed 
409 
transplant are retained through the duration of follow up.  
410 
 
411 
5.8. TERMINATION OF STUDY 
412 
The DSMC can recommend to terminate or temporarily suspend the clinical trial at any time 
413 
point. Halting guidelines are described in Section 9.4. Should the study be terminated or 
414 
suspended, the investigators, FDA, and IRB will be notified. The research staff and the IRB will 
415 
determine the best method to notify participants of study termination or suspension to ensure 
416 
participant safety, confidentiality, and data integrity. The investigators will notify all participants 
417 
of the termination or suspension and the reasons for such action. 
418 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
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6. STUDY PROCEDURES, MEASUREMENTS, AND VISIT SCHEDULE 
419 
6.1. CALEC MANUFACTURING 
420 
Details of CALEC manufacturing have been provided in the Manual of Procedures Section 7.1. 
421 
 
422 
6.2. PREPARATION AND ADMINISTRATION OF STUDY TREATMENTS 
423 
Biopsies and reconstruction CALEC procedures will be performed by the study PI, Dr. Ula 
424 
Jurkunas, to minimize surgeon variability. In extraneous circumstances where Dr. Ula Jurkunas 
425 
is not available to perform CALEC biopsy and/or reconstruction procedure, the study co-
426 
investigators Dr. Reza Dana and Dr. Jia Yin will perform the procedures.   
427 
 
428 
6.2.1. PREPARATION (CALEC) 
429 
6.2.1.1. LIMBAL BIOPSY 
430 
Two days prior to biopsy of the donor eye, all participants will be started on a topical 
431 
fluoroquinolone. Some participants may also start on vancomycin in the eye to be biopsied if the 
432 
participants are Methicillin-resistant Staphylococcus aureus (MRSA) positive or are considered 
433 
to be part of high-risk populations (e.g., health care personnel). 
434 
 
435 
6.2.1.2. TRANSPLANTATION 
436 
Two days prior to transplantation, the participant will be started on a topical fluoroquinolone (all 
437 
participants) and potentially vancomycin drops (in participants that are MRSA positive or in high-
438 
risk populations, e.g., health care personnel) in the recipient eye. 
439 
 
440 
6.2.2. ADMINISTRATION 
441 
6.2.2.1. CALEC PROCEDURE – BIOPSY 
442 
1. Participant will be taken to the preoperative area of the ambulatory surgery center where 
443 
standard operating procedures (SOP) will be employed in preparing the donor eye for the 
444 
surgery. 
445 
2. Type of anesthesia will be decided depending upon the age and overall functioning of the 
446 
participant. If the procedure is to be performed under general anesthesia (GA), GA consent 
447 
will be obtained from the participant or guardian. Otherwise, monitored intravenous 
448 
anesthesia (MIVA) will be performed after the consent. 
449 
3. The eye that is to be biopsied will be marked as such and the other eye will be covered. 
450 
4. Fluoroquinolone and proparacaine drops will be administered 3 times prior to the procedure. 
451 
5. Lidocaine 1% gel will be administered into the eye and the eye will be closed with tape. 
452 
6. The participant will be brought to the operating suite and positioned in a supine position 
453 
under the operating microscope in the manner typical for ophthalmic surgery.  
454 
7. The operative eye will be cleaned using 5% Betadine solution per standard surgical 
455 
protocol. Both the cul-de-sac and the eyelashes will be cleaned. 
456 
8. Under sterile conditions, a limbal biopsy of 3mm-by-3 mm (1 clock hour) will be dissected 
457 
from superior or inferior portion of the eye, at the discretion of the operating surgeon. The 
458 
actual size of the graft will be measured and captured for data collection. 
459 
9. The biopsied material will be placed into the container with HypoThermosol® FRS for 
460 
transfer to the Dana-Farber Cancer Institute. 
461 
10. The biopsied site in the conjunctiva will be closed using interrupted sutures and/or fibrin glue 
462 
per the surgeon’s discretion.. 
463 
11. Maxitrol or Tobradex ointment or drops will be placed onto the eye, if patient has no known 
464 
allergy. 
465 
12. Either a patch or shield will be placed over the eye. 
466 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 18 of 51 
6.2.2.2. CALEC PROCEDURE – TRANSPLANTATION 
467 
1. The participant will be taken to the preoperative area of the ambulatory surgery center 
468 
where standard operating procedures (SOP) will be employed in preparing the recipient eye 
469 
for the surgery. 
470 
2. Type of anesthesia will be decided depending upon the age and overall functioning of the 
471 
participant. If the procedure is to be performed under general anesthesia (GA), GA consent 
472 
will be obtained from the participant or guardian. Otherwise, monitored intravenous 
473 
anesthesia (MIVA) will be performed after the consent. 
474 
3. In cases of MIVA anesthesia, peribulbar block will be injected in the operated eye. 
475 
4. The recipient eye that is to be operated on (i.e., the eye with LSCD) will be marked as such 
476 
and the other eye will be covered. 
477 
5. Fluoroquinolone and proparacaine drops will be administered 3 times prior to the procedure. 
478 
6. Peribulbar or retrobulbar block with 50:50 mixture of lidocaine and bupivacaine will be 
479 
injected into the recipient eye. 
480 
7. The participant will be brought to the operating suite and positioned in a supine position 
481 
under the operating microscope in the manner typical for ophthalmic surgery. 
482 
8. The operative eye will be cleaned using 5% Betadine solution per standard surgical 
483 
protocol. Both the cul-de-sac and the eyelashes will be cleaned. 
484 
9. If excessive ocular surface bleeding is detected, topical epinephrine (1:10,000) will be used 
485 
to constrict the blood vessels and minimize bleeding prior to or during the procedure. A 
486 
conjunctival peritomy may be performed per investigator discretion. The fibrovascular tissue 
487 
will be dissected from the limbus and the cornea. Hemostasis will be achieved by wet-field 
488 
cautery. Mitomycin C may be placed underneath the conjunctiva per the surgeon’s 
489 
discretion. 
490 
10. The transwell with CALEC graft inside will be removed from the original container and will be 
491 
placed on a sterile silicone platform and rinsed with BSS® Sterile Irrigation Solution. A free-
492 
held trephine, size depending on the operated eye, will be used to punch the graft. The size 
493 
of trephine used will be recorded in the operative note. The transwell with the remnants of 
494 
the graft will be lifted off. The trephined CALEC graft on the transwell membrane will be 
495 
lifted with the forceps and transferred to the surgical field where the CALEC graft will be 
496 
peeled from the transwell membrane and centered onto the ocular surface with epithelium 
497 
side up. CALEC will be secured with sutures and/or fibrin glue per the surgeon’s discretion. 
498 
The conjunctiva will be closed using sutures and/or fibrin glue per the surgeon’s discretion. 
499 
At the end of the procedure, fluorescein will be used to assess epithelial integrity. Lack of 
500 
fluorescein uptake in the central cornea will indicate that epithelium of the CALEC graft is 
501 
intact. 
502 
11. Bandage contact lens will be placed over the graft. 
503 
12. Subconjunctival injection of Kefzol and Decadron will be given, if patient has no known 
504 
allergy. 
505 
13. Maxitrol or Tobradex ointment or drops will be placed onto the eye, if patient has no known 
506 
allergy. 
507 
14. A patch and/or shield will be placed over the eye. 
508 
 
509 
6.3. MODIFICATION OF STUDY INTERVENTION FOR A PARTICIPANT 
510 
If a participant has an allergy to any of the medications listed in the protocol, that medication will 
511 
not be used and/or will be substituted with an appropriate alternative based on the investigators’ 
512 
discretion. If participant has a latex allergy, standard precautions will be employed to avoid latex 
513 
products during the peri- and intra-operative periods. 
514 
 
515 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 19 of 51 
6.4. ADDITIONAL SURGERIES 
516 
If a participant requires any additional surgeries for visual rehabilitation in the recipient eye, 
517 
including cataract surgery or corneal transplantation, the surgeries will be performed at least 3 
518 
months after corneal reconstruction with CALEC. 
519 
 
520 
6.5. MEDICATIONS AND TREATMENTS 
 
521 
6.5.1.  CONCOMITANT MEDICATION/TREATMENTS 
522 
Participants who enter the study taking anti-glaucoma medication will be able to continue that 
523 
medication throughout the duration of the study. If participants develop increased intraocular 
524 
pressure (IOP) during the study, anti-glaucoma medications may be prescribed that include one 
525 
or more or in combination formulations. 
526 
 
527 
6.5.2.  ACCEPTABLE CONCOMITANT MEDICATIONS  
528 
Participants will be able to use anti-glaucoma medications. Participants will be able to have 
529 
increased dose of topical corticosteroids throughout the course of the study if determined to be 
530 
necessary by the treating ophthalmologist. Participants who develop infections will be treated 
531 
with appropriate antimicrobial agents such as antibiotics, anti-viral and/or anti-fungal agents. 
532 
Participants will be allowed to use ointments that have antibiotic and/or steroid components.  
533 
 
534 
Whenever possible, non-preserved medications will be prescribed to reduce epithelial toxicity.  
535 
 
536 
Current medication regimens will not be changed, if possible. 
537 
 
538 
6.5.3.  PROHIBITED CONCOMITANT MEDICATIONS  
539 
• 
Other investigational treatments 
540 
 
541 
6.5.4.  STUDY MEDICATION REGIMEN 
542 
The pre-study topical ocular medication regimens of all participants will be reviewed at study 
543 
enrollment, and may be changed based on investigator’s clinical judgement. All participants will 
544 
be prescribed a topical fluoroquinolone, a topical steroid 1%, Vancomycin 14 mg/mL (see 
545 
below), 20% autologous serum eye drops, and artificial tears in both eyes as follows. If 
546 
participants have or develop an allergy to the drops, the investigators will prescribe an 
547 
appropriate substitute.  The regimen applies to only those participants who complete the biopsy 
548 
and/or the transplant, as applicable.  
549 
 
550 
Screening Visit 
551 
If the conjunctival culture taken at screening reveals the presence of MRSA bacteria or the 
552 
participant is in a high-risk population for MRSA (i.e. hospital employee), the participant will be 
553 
treated with Vancomycin 14 mg/mL, four times per day in both eyes. 
554 
 
555 
Day 1 Post-Biopsy Visit 
556 
The participant will start a topical fluoroquinolone and  a topical steroid 1% in the donor eye on 
557 
Day 1 after biopsy until time of corneal reconstruction (approximately two weeks).  The topical 
558 
steroid will be started at four times per day and adjusted based on clinical judgement. If 
559 
previously using Vancomycin to treat MRSA, the participant will continue using it four times per 
560 
day in both eyes. 
561 
 
 
562 
Day 1 Post-Transplant Visit (Corneal Reconstruction) 
563 
• 
The participant will start a topical fluoroquinolone and a topical steroid 1% in recipient eye. 
564 
S/he will continue regimen until 2-week visit. If previously using Vancomycin to treat MRSA, 
565 
the participant will continue using four times per day in both eyes. 
566 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 20 of 51 
• 
The participant will start 20% autologous serum eye drops in the recipient eye after corneal 
567 
reconstruction and will continue it for at least 1 month. Dosage will be adjusted based on 
568 
clinical judgement. In addition the participant will start artificial tears four times per day in 
569 
both eyes and continue throughout the course of the study. 
570 
 
571 
1-Week Visit after Transplant 
572 
The participant will continue all medications as prescribed at Day 1 Post-Graft visit.  Dosage will 
573 
be adjusted based on clinical judgement. 
574 
 
575 
2-Week Visit after Transplant 
576 
• 
The participant may start taper of topical fluoroquinolone and topical steroid 1% in the 
577 
recipient eye; changes to dosage will be made for individual participants based on clinical 
578 
judgement. The participant will be able to stop the steroid and fluoroquinolone in the donor 
579 
eye, unless inflammation persists in which case usage will continue up to 4 weeks. 
580 
• 
The participant will continue autologous serum eye drops and artificial tear use four times 
581 
per day in both eyes. Vancomycin will be tapered according to the same schedule as the 
582 
fluoroquinolone and the topical steroid 1%. 
583 
 
584 
1-Month Visit after Transplant 
585 
The participant will continue all medications as described at the 2-week visit. Around the time of 
586 
this visit, participants will be starting their daily dose of the topical fluoroquinolone and the 
587 
topical steroid 1% as described in the taper regimen at the 2-week visit. If the participant was 
588 
previously using Vancomycin, it will be decreased along the same schedule. The autologous 
589 
serum eye drops may be stopped at investigator discretion based on participant symptoms of 
590 
discomfort and epithelial healing. 
591 
 
592 
3-Month Visit after Transplant 
593 
From this point forward, the participant’s medication regimens will no longer follow a protocol-
594 
dependent schedule and will be adjusted as medically necessary. Participants who have been 
595 
MRSA positive will undergo another conjunctival culture and Vancomycin dosing will be 
596 
adjusted based on findings. If participant has to continue bandage contact lens, as determined 
597 
by investigator discretion based on participant symptoms of discomfort and epithelial healing, 
598 
the fluoroquinolone will be continued for the duration of lens wear. 
599 
 
600 
6.5.5. OTHER TREATMENTS 
601 
At least one ocular punctal plug will be placed in each eye as needed for which the Schirmer’s 
602 
Test is below 10 mm and the patient does not have an existing plug or had punctual cautery in 
603 
the past. The plug(s) will be placed at the screening visit and then replaced at subsequent visits 
604 
if they fall out. Punctal plugs may also be initiated at any visit per usual clinical practice. A 
605 
bandage contact lens will be placed on the treated eye of all subjects up until 30 days post 
606 
procedure. The bandage contact lens may continue to be worn past 30 days post procedure if 
607 
there is evidence of epithelial defect or if deemed necessary by the treating physician.  
608 
 
609 
6.6. CLINICAL EVALUATIONS  
610 
The following is a summary of how each clinical evaluation will be performed.  Section 6.8 
611 
summarizes the study visit schedule and all assessments performed at each visit.  The MOP 
612 
provides more details regarding the order of testing and by whom each assessment is 
613 
performed.  
614 
 
615 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 21 of 51 
6.6.1.  SYMPTOM ASSESSMENT 
616 
The Ocular Surface Disease Index (OSDI) and Symptom Assessment iN Dry Eye (SANDE) 
617 
questionnaires will be administered by certified study personnel prior to initiating the ophthalmic 
618 
exam.  
619 
 
620 
6.6.1.1. OCULAR SURFACE DISEASE INDEX (OSDI) 
621 
This disease-specific questionnaire includes three subscales: ocular discomfort (OSDI 
622 
symptoms), which includes symptoms such as gritty or painful eyes; functioning (OSDI-
623 
function), which measures limitation in performance of common activities such as reading and 
624 
working on a computer; and environmental triggers (OSDI-triggers), which measures the impact 
625 
of environmental triggers, such as wind or drafts, on dry eye symptoms (Manual of Procedure 
626 
(MOP), Section 21, Vitale et al., 2004).   
627 
 
628 
6.6.1.2. SYMPTOM ASSESSMENT IN DRY EYE (SANDE) 
629 
The SANDE questionnaire uses a horizontal visual analog technique to quantify each 
630 
participant’s symptomatology with regard to dryness and/or irritation (MOP, Section 22). Each 
631 
index will use a 100mm line to individually assess both the average frequency and the average 
632 
severity of symptoms of ocular discomfort or dryness experienced by the participant. The 
633 
participant will be asked to put a mark on two given lines to depict the extent of their symptoms 
634 
separately in terms of frequency and severity – the mark will be measured and recorded by the 
635 
study team. 
636 
 
637 
6.6.2.  VISUAL ACUITY 
638 
At each visit, participants’ best corrected visual acuity (BCVA) of both eyes will be assessed by 
639 
Snellen visual acuity and recorded (MOP, Section 23). Manifest refraction will be performed if 
640 
pinhole improves BCVA more than 1 line.  
641 
 
642 
6.6.3. INTRAOCULAR PRESSURE (IOP) EVALUATION 
643 
IOP will be evaluated by one or a combination  of the available modalities noted below 
644 
depending on the cooperation of the participant and ocular appearance on the exam as well as 
645 
ability to obtain the reading (MOP, Section 24). 
646 
• 
Tonopen: To measure IOP using tonopen applanation, the Tono-Pen XL (Mentor, Santa 
647 
Barbara, CA) will be calibrated daily. Following administration of 0.5% proparacaine 
648 
hydrochloride in the cul-de-sac, the pen tip (with disposable cover) will be touched to the 
649 
central cornea until a reading is measure. Only measurements with a 5% standard error 
650 
will be accepted. If error is greater than 5%, the measurement will be repeated. 
651 
• 
Pneumotonometry: Pneumotonometer (Reichart) measurements will be taken 
652 
perpendicularly to the cornea, at the center of the cornea after calibration according to 
653 
the manufacturer’s instructions in the event Tonopen is unable to provide a reading 
654 
• 
Palpation: The IOP of the donor eye will be determined by palpation for one week post 
655 
biopsy.  The IOP of the recipient eye will be determined by palpation until the thirteen 
656 
week visit after transplant. 
657 
 
658 
6.6.4. SLIT LAMP EXAMINATION 
659 
At each visit, participants will undergo assessment of eyelid, conjunctiva, cornea, and all 
660 
intraocular structures of both eyes, by a certified study investigator who was not the investigator 
661 
performing the biopsy or transplant for that participant. This may include a research associate or 
662 
research fellow who does not perform surgeries but functions as a clinical examiner investigator 
663 
only.  The number of such investigators will be limited and each will complete sample 
664 
assessments on the primary efficacy endpoints (both the corneal epithelial defect surface area 
665 
and NEI scale gradings noted below) as part of their study training and certification.   
666 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
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Page 22 of 51 
 
667 
6.6.4.1. CORNEAL OPACIFICATION 
668 
The cornea will be examined and its opacification will be graded according to the Fantes Scale 
669 
(MOP, Section 26) (Fantes et al., 1990). 
670 
 
671 
6.6.4.2. CORNEAL FLUORESCEIN STAINING  
672 
For the clinical exam with corneal fluorescein staining, a single Akorn FUL-GLO Fluorescein 
673 
Sodium strip will be wetted with a drop of sterile saline and applied to the inferior fornix and 
674 
examination and photography (Section 6.6.7.2 below) will be performed between 2-5 minutes 
675 
after the instillation of fluorescein.   
676 
 
677 
The entire cornea will be examined using slit lamp evaluation with a yellow barrier filter (#12 
678 
Wratten) and cobalt blue illumination (staining is more intense when it is observed with a yellow 
679 
filter). Staining with fluorescein will be used to determine presence of corneal epithelial defects. 
680 
The extent of corneal fluorescein staining will be evaluated using the National Eye Institute 
681 
(NEI) grading scale (MOP, Section 25). Each of five corneal zones (superior, nasal, central, 
682 
inferior, and temporal) will be graded from 0 (normal) to 3 (severe) and staining score of the 
683 
cornea will range from 0 and 15 points. Epithelial defects (as defined as confluent epithelial 
684 
staining of >1 mm2) will be measured at the greatest horizontal and vertical dimensions, and 
685 
surface area will be calculated in mm2.   
686 
 
687 
6.6.5. SCHIRMER’S TEST 
688 
The Schirmer’s test will be performed with anesthesia by placing a narrow filter-paper strip 
689 
(5mm × 35mm strip of Whatman #41 filter paper) in the inferior cul-de-sac (MOP, Section 27). 
690 
This test is to be conducted in a dimly lit room. The participant will be instructed to gently close 
691 
their eyes until five minutes have elapsed. The strips will be removed. Since the tear front will 
692 
continue advancing a few millimeters after it has been removed from the eyes, it is important to 
693 
mark the tear front with a ball-point pen at precisely five minutes. Aqueous tear production will 
694 
be measured by the length in millimeters that the strip wets in 5 minutes.  
695 
 
696 
6.6.6. IMPRESSION CYTOLOGY 
697 
At 12, 15, and 18 months after transplantation, if there is clinical suspicion of limbal stem cell 
698 
deficiency in the recipient eye, an impression cytology of the ocular surface will be performed to 
699 
confirm the diagnosis. Impression cytology will be performed in the recipient eye after 
700 
application of the topical anesthetic. Nitrocellulose membranes will be firmly pressed onto the 
701 
ocular surface (as shown in MOP) for 5 seconds. Each membrane will cross the limbus so as to 
702 
collect cells from the cornea, the limbus, and the conjunctiva. The membranes will then be 
703 
removed and fixed in methanol solution immediately. Samples will then be taken to the 
704 
pathology laboratory for further staining and analysis. 
705 
 
706 
6.6.7. SLIT LAMP PHOTOGRAPHY 
707 
Digital corneal photography to measure neovascular area and epithelial defect area will be done 
708 
by a certified study photographer or research associate, using a slit lamp with a digital camera 
709 
attachment and a flash-through-the-slit illumination system. 
710 
 
711 
6.6.7.1. PHOTOGRAPHY FOR NEOVASCULAR AREA 
712 
The entire cornea will be pictured using diffuse illumination and 10x magnification. If lids are 
713 
drooping, the photographer will attempt to gently remove them from area of focus with a cotton 
714 
swab. Participants with dark irises, which may prohibit a clear image, may be dilated with 
715 
tropicamide only (without use of an adrenergic agent that may induce vascular ‘blanching’) to 
716 
enhance visualization of corneal blood vessels. Digital photographs will be taken and the 
717 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
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Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 23 of 51 
images will be uploaded into the graphics editing software (ImageJ). The neovascular area will 
718 
be calculated as described in MOP. Digital slit lamp corneal pictures will be analyzed using 
719 
graphics-editing software (Photoshop) and a mathematical program (Matlab script). After the 
720 
total area is delineated, the blood vessels will be isolated using Photoshop. Neovascular area 
721 
(in pixels) is computed using a Matlab script to assist a qualified technician to measure the area 
722 
of the vessels themselves. 
723 
 
724 
6.6.7.2. PHOTOGRAPHY FOR EPITHELIAL DEFECT AREA 
725 
For photography with corneal fluorescein staining, a single Akorn FUL-GLO Fluorescein Sodium 
726 
strip will be wetted with a drop of sterile saline and applied to the inferior fornix and examination 
727 
(Section 6.6.4.2 above) and photography will be performed between 2-5 minutes after the 
728 
instillation of fluorescein.   
729 
 
730 
Following staining, the entire cornea will be imaged using cobalt blue filter of the slit lamp. A 
731 
magnification of 10x with diffuse illumination will be used. Digital photographs will be taken and 
732 
the images will be uploaded into the graphics editing software. The epithelial defect area will be 
733 
calculated. Digital slit lamp corneal pictures will be analyzed using ImageJ. The total area of the 
734 
epithelial defect(s) will be measured in pixels. Then, total corneal area will also be measured in 
735 
pixels. Then, the ratio of the total corneal area which is covered by epithelial defect(s) will be 
736 
calculated in percentage.  
737 
 
738 
6.6.8. LID MARGIN AND CONJUNCTIVA CULTURES 
739 
Conjunctival cultures will be performed to evaluate the microbiologic colonization at the 
740 
screening visit in all participants and at the 3- and 12-month visits only if MRSA was detected at 
741 
screening. A moistened swab will be used to get specimen from the lower fornix in each eye, 
742 
and will be inoculated above the “R” and “L” on the blood and chocolate plates in the shape of 
743 
“C.” The plates will be placed in an incubator set to 35°C with 5% CO2. All plates will be 
744 
observed daily for seven days for the formation of microbial colonies. The bacteria will be 
745 
identified by using standard microbiologic techniques. 
746 
 
747 
6.6.9. FUNDUS EXAMINATION 
748 
A dilated fundoscopy (fundus examination) will be performed to evaluate the fundus for 
749 
abnormalities. If the fundoscopy cannot be performed, a B-scan ultrasound will be performed 
750 
instead to evaluate the fundus for abnormalities.    
751 
 
752 
6.6.10 ANTERIOR SEGMENT OPTICAL COHERENCE TOMOGRAPHY 
753 
Anterior segment optical coherence tomography (AS-OCT) is a non-contact imaging modality 
754 
that provides high-resolution cross-sectional images of ocular structures. The subject will be 
755 
positioned in front of the AS-OCT machine (RTVue-100, OptoVue, Freemont, CA). One scan of 
756 
the central cornea and one scan of the limbus in each quadrant (superior, inferior, nasal, and 
757 
temporal) will be obtained using an automated scanning algorithm. AS-OCT imaging of both 
758 
eyes will be performed at the baseline, 12 and 18-month post-operative visit. Central epithelial 
759 
thickness is calculated by an automatic algorithm. The deepest limbal epithelial thickness in 
760 
each quadrant will be measured manually on cross-sectional images. The outcomes are central 
761 
corneal epithelial thickness and limbal epithelial thickness in superior, inferior, nasal, and 
762 
temporal quadrants. There may be future measurements using these images that are currently 
763 
unknown. AS-OCT will be done by a certified study photographer or research associate. 
764 
 
765 
6.6.11 IN VIVO CONFOCAL MICROSCOPY  
766 
In vivo confocal microscopy (IVCM) is an imaging method that allows visualization of the corneal 
767 
structures at the cellular level. A drop of topical anesthetic will be instilled in each eye prior to 
768 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 24 of 51 
the procedure and a temporary bandage contact lens may be applied prior to imaging for patient 
769 
comfort and removed after imaging. One drop of hypromellose 2.5% is applied to the patient’s 
770 
eye and one drop of hypromellose 0.3% will be placed on the objective lens of the microscope 
771 
according to the manufacturer’s instructions.  IVCM will be performed on each eye with a 
772 
Heidleberg retina tomograph (HRTII)/Rostock cornea module (RCM) (Heidelberg Engineering 
773 
GmbH, Dossenheim, Germany) with a water-immersion objective lens. One scan of the central 
774 
cornea and one scan of each quadrant of the limbus (superior, nasal, temporal, and inferior) will 
775 
be obtained. IVCM of both eyes will be performed at the baseline visit, 12 and 18-month post-
776 
operative visits unless patient reports eye pain, discomfort, irritation and cannot tolerate the 
777 
procedure, or there is clinical contraindication in the case of corneal epithelial defect, 
778 
hemorrhage, or inflammation. Images will be analyzed to determine the presence or absence of 
779 
the following cell types: corneal epithelial cells are defined as polygonal cells with bright, well-
780 
defined borders, dark cytoplasm and no visible nuclei; conjunctival cells are defined as cells with 
781 
bright nuclei and ill-defined borders.  Limbal palisades of Vogt are defined as hyper-reflective 
782 
double contoured linear structures that alternate with islands of epithelial cells. The outcomes 
783 
are: presence (or absence) of corneal epithelial cells in the central cornea and limbus, presence 
784 
(or absence) of conjunctival epithelial cells in the central cornea and limbus, and the presence 
785 
(or absence) of limbal palisades of Vogt. There may be future analysis of the images that are 
786 
currently unknown.  IVCM will be done by a certified study photographer or research associate. 
787 
 
788 
6.7. LABORATORY EVALUATIONS 
789 
Participants will have blood drawn at the Massachusetts Eye and Ear Infirmary Clinical 
790 
Laboratory at screening (see list of tests run at screening).An additional blood draw will be 
791 
collected within 7 days before or after biopsy to test for Hepatitis B, Hepatitis C, and HIV, and 
792 
results communicated to CMCF prior to release of the product. 
793 
 
794 
Additionally, female participants of child-bearing potential will undergo a urine pregnancy test at 
795 
screening for study eligibility determination and then again at the visit before CALEC 
796 
transplantation. 
797 
 
798 
Participants with uncontrolled diabetes, as defined by the most recent HbA1c >8.5% (at the 
799 
screening visit or within 3 months prior to screening visit) and with decreased renal function, as 
800 
defined by eGFR (estimated glomerular filtration rate) below 60 mL/min per 1.73 m2, will be 
801 
excluded from the study. Similarly, we will check lab values for liver enzymes, hemoglobin, 
802 
platelets, prothrombin time, partial thromboplastin time, and international normalized ratio. 
803 
Aspartate aminotransferase or alanine aminotransferase levels >3x institutional upper limit of 
804 
normal will be considered abnormal. Total bilirubin >2.0x institutional upper limit of normal 
805 
(except participants with known Gilbert’s syndrome) will be considered abnormal. Further, 
806 
platelet levels <100,000 or >450,000 per microliter, and male hemoglobin of <11.0 g/dL, and 
807 
female hemoglobin levels of <10.0 g/dL, will also be consider abnormal. Finally, prothrombin 
808 
time >16 seconds or activated partial thromboplastin time >35 seconds in participants not taking 
809 
warfarin, and an international normalized ratio >3 in participants taking warfarin, will be 
810 
considered abnormal. Participants testing positive for HIV or AIDS or active hepatitis B or C will 
811 
also be excluded. If any of these lab values are found to be abnormal, the participants will be 
812 
excluded and referred to their primary care physicians for further workup and treatment. 
813 
 
814 
6.8. STUDY SCHEDULE 
815 
Below is a summary of the study visit schedule.  The following sections summarize all 
816 
assessments performed at each visit.  The MOP provides more details regarding the order of 
817 
testing. For a summary table of study visit schedule and all assessments performed, see 
818 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 25 of 51 
Appendix VII. All visits occur after the participant has been enrolled (after obtaining informed 
819 
consent). 
820 
 
821 
• 
Screening Visit  
822 
• 
Baseline Visit –required within 30 days of Screening Visit 
823 
• 
Biopsy - required within 25 days of Baseline Visit 
824 
• 
Post-Biopsy Visit – required 1 day after biopsy 
825 
• 
Pre-operative Visit - required 1 to 5 days prior to transplant 
826 
• 
Transplant – anticipated to occur 10 to 30 days after biopsy    
827 
• 
Day 1, Week 1, Week 2*, and Month 1 Visits  
828 
o Same for Phase A and B participants 
829 
 
Only required for participants who complete a transplant  
830 
 
Visit schedule is timed from transplant date  
831 
• 
Month 3, 6, 9*, 12, 15*, and 18 Visits 
832 
o Same for Phase A and B participants 
833 
 
Only required for participants who complete a transplant  
834 
 
Visit schedule is timed from transplant date  
835 
 
836 
* The 2 week, 9 month, and 15 month visits will be optional for participants for whom travel is 
837 
prohibitive.  For participants who opt out of these visits, the clinical site will work with the 
838 
participant’s usual care ophthalmologist on the following:   
839 
(1) Advise that the participant follows the protocol’s post-operative medication regimen, 
840 
although the regimen will not be enforced by the protocol in these participants. 
841 
(2) Request adverse events be reported to the clinical site, under a medical release from 
842 
the participant.   
843 
 
844 
 
845 
6.8.1.  STUDY SCREENING  
846 
Prospective participants, as defined by the inclusion/exclusion criteria, will be considered for 
847 
entry into this study. The study design and treatment regimen will be discussed with each 
848 
participant. Written informed consent will be obtained before any study-specific screening 
849 
evaluations are performed. The following evaluations and procedures will be performed for all 
850 
participants during the screening period: 
851 
 
852 
• 
Informed consent process culminating in signed consent document 
853 
• 
Eligibility Assessment 
854 
• 
Record current ocular and systemic medications  
855 
• 
Record medical/surgical history in the past 5 years 
856 
• 
Record demographic data, including date of birth, sex, and race/ethnicity 
857 
• 
Review of systems 
858 
• 
Blood draw for testing for confirmation of eligibility criteria, including: 
859 
o Hepatitis B 
860 
o Hepatitis C 
861 
o HIV, AIDS 
862 
o CBC (including platelet and hemoglobin levels) 
863 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 26 of 51 
o eGFR 
864 
o HbA1c, if needed (does not need to be repeated at screening visit if done 
865 
within the last 3 months prior to screening visit) 
866 
o Aspartate aminotransferase and alanine aminotransferase levels 
867 
o Bilirubin levels 
868 
o Prothrombin and thromboplastin time  
869 
• 
Urine pregnancy test for women of childbearing potential 
870 
• 
Slit lamp examination (both eyes), including 
871 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
872 
o Staining with fluorescein (extent of staining and epithelial defect area) 
873 
(both eyes) 
 
874 
• 
Visual Acuity BCVA (both eyes) 
875 
• 
Conjunctival swab and culture (both eyes) 
876 
• 
Intraocular pressure (both eyes) 
877 
• 
Fundus examination or B-scan ultrasound (both eyes) 
878 
• 
Slit lamp photographs (both eyes) by certified technician, including 
879 
o Photography for neovascular area 
880 
o Photography (with fluorescein) for epithelial defect 
881 
• 
Schirmer’s test with anesthesia (both eyes) 
882 
• 
Referral to primary care physician to confirm general health prior to surgery, as needed 
883 
according to MEEI hospital SOPs 
884 
• 
Punctal plugs, if needed (Section 6.5.5) (both eyes) 
885 
 
886 
In addition, blood draw will be performed for autologous serum eye drops prior to transplantation 
887 
per CMCF guidelines. 
888 
 
889 
6.8.2. BASELINE (WITHIN 30 DAYS OF SCREENING VISIT) 
890 
A baseline visit will be conducted after a participant is considered eligible to participate as 
891 
determined by the screening visit. Baseline visit procedures must occur within 30 days after the 
892 
screening visit 
893 
 
 
894 
Baseline procedures may be conducted over multiple visits after screening. 
895 
 
896 
The following procedures will be conducted for the baseline visit: 
897 
 
898 
• 
Review of pre-operative health screening results to confirm general health prior to surgery 
899 
according to MEEI hospital SOPs  
900 
• 
Slit lamp examination (both eyes), including 
901 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
902 
o Staining with fluorescein (extent of staining and epithelial defect area) (both eyes) 
903 
• 
Visual acuity BCVA (both eyes) 
904 
• 
Intraocular pressure (both eyes) 
905 
• 
Slit lamp photographs (both eyes) by certified technician, including 
906 
o Photography for neovascular area 
907 
o Photography (with fluorescein) for epithelial defect 
908 
• 
Anterior Segment Optical Coherence Tomography 
909 
• 
In Vivo Confocal Microscopy 
910 
• 
Punctal plugs, if needed (Section 6.5.5) (recipient eye only) 
911 
• 
Assessment of changes in medical conditions since screening  
912 
• 
Symptom assessment 
913 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 27 of 51 
 
914 
 
915 
In addition, within seven days before or after limbal biopsy, blood draw will be performed for 
916 
donor serology testing in CALEC participants only. 
917 
 
918 
6.8.3. LIMBAL BIOPSY (WITHIN 25 DAYS OF BASELINE VISIT) 
919 
Following the baseline visit, a biopsy of the donor eye will be taken as an outpatient procedure 
920 
for all participants receiving the CALEC. The biopsy must occur within 25 days after the 
921 
baseline visit. Refer to section 6.2. 
922 
 
923 
6.8.4. TRANSPORTATION OF TISSUE TO CMCF & PREPARATION OF CALEC 
924 
If participants are enrolled in the CALEC arm, the tissue taken from the donor eye during the 
925 
biopsy will be taken to Connell and O’Reilly Families Cell Manipulation Core Facility (CMCF) at 
926 
the Dana-Farber Cancer Institute (DFCI) per operating procedures (MOP, Section 7).  
927 
 
928 
6.8.5. POST-BIOPSY ASSESSMENT OF DONOR EYE (1-2 DAYS AFTER CALEC BIOPSY) 
929 
• 
Slit lamp examination (donor eye) including 
930 
o Staining with fluorescein (extent of staining and epithelial defect area)  
931 
• 
Visual acuity  (BCVA) (donor eye) 
932 
• 
Intraocular pressure (donor eye) 
933 
• 
Adverse event assessment   
934 
• 
Slit lamp photos (donor eye) by certified technician, including: 
935 
o Photography (with fluorescein) for epithelial defect 
936 
• 
Punctal plug placement, if needed (Section 6.5.5) 
937 
 
938 
6.8.6. TRANSPORTATION OF CALEC FROM CMCF TO MEEI 
939 
The CALEC is anticipated to be released 10 to 30 days after biopsy, upon which it will be 
940 
transported from CMCF to MEEI (MOP, Sections 7.1.3 and 7.1.4). 
941 
 
942 
6.8.7. PREOPERATIVE ASSESSMENT OF RECIPIENT EYE (1 TO 5 DAYS PRIOR TO 
943 
TRANSPLANT)  
944 
A preoperative assessment will be evaluated prior to reconstruction to confirm fitness for 
945 
reconstruction of the recipient eye. All participants will receive this preoperative assessment 1 to 
946 
5 days prior to the day of corneal reconstruction (transplant). 
947 
 
948 
The following procedures will be performed at these visits: 
949 
• 
Urine pregnancy test for women of childbearing potential 
950 
• 
Slit lamp examination (recipient eye) including  
951 
o Corneal opacification (as defined by Fantes Scale) 
952 
o Staining with fluorescein (extent of staining and epithelial defect area) 
953 
• 
Visual acuity BCVA (recipient eye) 
954 
• 
Intraocular pressure (recipient eye) 
955 
• 
Adverse event assessment 
956 
• 
Punctal plugs, if needed (Section 6.5.5) (both eyes) 
957 
 
958 
Participants will be asked to report any pregnancies that occur within three days prior to 
959 
surgery. 
960 
 
961 
6.8.8. CORNEAL RECONSTRUCTION WITH CALEC (DAY 0)  
962 
Participants will undergo corneal reconstruction within 24 hours of release of CALEC.   
963 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 28 of 51 
 
964 
A detailed description of the procedures can be found in Section 6.2.2. All participants will be 
965 
discharged the day of surgery, unless unforeseen complications prohibit discharge. If a 
966 
participant is admitted for observation, the Day 1 post-operative visit will occur while the 
967 
participant is in the hospital. All other visits will follow as originally scheduled. 
968 
 
969 
6.8.9. FOLLOW-UP PERIOD 
970 
Each participant will be followed for a period of 18 months from the time of the transplantation. 
971 
 
972 
Participants will be asked to report any pregnancies that occur within seven days after 
973 
transplant. 
974 
 
975 
6.8.10. POST-TRANSPLANT (DAY 1) 
976 
• 
Slit lamp examination (both eyes) including 
977 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
978 
o Staining with fluorescein (extent of staining and epithelial defect area) (donor eye 
979 
required; recipient eye at discretion of investigator ) 
980 
• 
Visual acuity  (BCVA)(both eyes) 
981 
• 
Intraocular pressure (both eyes) 
982 
• 
Slit lamp photographs (both eyes) by certified technician, including 
983 
o Photography for neovascular area 
984 
o Photography (with fluorescein) for epithelial defect (donor eye required; recipient 
985 
eye at discretion of clinician) 
986 
• 
Punctal plugs, if needed (Section 6.5.5) (both eyes) 
987 
• 
Application of bandage contact lens, if needed (Section 6.5.5) 
988 
• 
Adverse event assessment 
989 
 
990 
6.8.11. POST-OP FOLLOW-UP: WEEKS 1, 2, MONTH 1 (4 WEEKS ± 3 DAYS) AND 
991 
MONTHS 3, 6, 9, 12 (13, 26, 39, 52 WEEKS ± 1 WEEK), AND MONTHS 15, AND 18 (65 AND 
992 
78 WEEKS ±2 WEEKS) 
993 
• 
Symptom assessment (every visit except week 1, week 2, month 1) 
994 
• 
Slit lamp examination (both eyes) including 
995 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
996 
o Staining with fluorescein (extent of staining and epithelial defect area) (donor eye 
997 
required; recipient eye at discretion of investigator at week 1 and 2, and required at 
998 
month 1 and thereafter.)  
999 
• 
Visual acuity BCVA (both eyes) 
1000 
• 
Intraocular pressure (both eyes) 
1001 
• 
Slit lamp photographs (both eyes) by certified technician, including 
1002 
o Photography for neovascular area 
1003 
o Photography (with fluorescein) for epithelial defect (donor eye required; recipient eye 
1004 
at discretion of investigator at week 1 and 2, and required at month 1 and thereafter) 
1005 
• 
Anterior Segment Optical Coherence Tomography (months 12 and 18) 
1006 
• 
In Vivo Confocal Microscopy (months 12 and 18) 
1007 
• 
Punctal plugs, if needed (Section 6.5.5) 
1008 
• 
Application of bandage contact lens, if needed (Section 6.5.5) 
1009 
• 
Conjunctival swab and culture (months 3 and 12, only if MRSA was detected at screening 
1010 
visit) 
1011 
• 
Impression cytology (in the recipient eye will be done as needed to confirm clinically 
1012 
suspected LSCD months 12, 15, and 18)  
1013 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 29 of 51 
• 
Adverse event assessment 
1014 
 
1015 
6.8.12. EARLY TERMINATION VISIT 
1016 
Participants who withdraw from the study or are unable to complete all of the study visits will be 
1017 
asked to complete an early termination visit prior to discontinuing their participation. The early 
1018 
termination visit will include the following: 
1019 
 
1020 
• 
Symptom Assessment  
1021 
• 
Visual acuity  (BCVA) (both eyes) 
1022 
• 
Intraocular pressure (both eyes) 
1023 
• 
Slit lamp examination (both eyes) including 
1024 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
1025 
o Staining with fluorescein (extent of staining and epithelial defect area) (both eyes) 
1026 
• 
Slit lamp photographs (both eyes) by certified technician, including 
1027 
o Photography for neovascular area 
1028 
o Photography (with fluorescein) for epithelial defect 
1029 
• 
Punctal plugs, if needed (Section 6.5.5) 
1030 
• 
Bandage contact lens, if needed (Section 6.5.5) 
1031 
• 
Adverse event assessment 
1032 
 
1033 
6.8.13. UNSCHEDULED VISIT 
1034 
Should a study participant need to be seen for medical reasons at a time point outside of the 
1035 
study protocol, the visit will follow the Week 1 schedule. Assessments may include, but are not 
1036 
limited to: 
1037 
• 
Visual acuity BCVA (both eyes) 
1038 
• 
Intraocular pressure (both eyes) 
1039 
• 
Slit lamp examination (both eyes) including 
1040 
o Corneal opacification (as defined by Fantes Scale) (both eyes) 
1041 
o Staining with fluorescein (extent of staining and epithelial defect area) (each eye  at 
1042 
discretion of investigator) 
1043 
• 
Punctal plugs, if needed (Section 6.5.5) 
1044 
• 
Bandage contact lens, if needed (Section 6.5.5) 
1045 
• 
Adverse event assessment 
1046 
 
1047 
 
 
1048 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 30 of 51 
7. OUTCOME MEASURES 
1049 
The following is a summary of the definition of the outcome measures. The Statistical Analysis 
1050 
Plan details the analysis approach for all outcomes.   
1051 
 
1052 
7.1. PRIMARY OUTCOME MEASURES 
1053 
7.1.1. SAFETY MEASURES 
1054 
The occurrence of the following adverse events at any time during the 18 months of follow-up in 
1055 
the recipient eye will serve as the primary safety events of interest:   
1056 
 
1057 
1. Ocular infection (defined as endophthalmitis or microbial keratitis [bacterial, fungal, 
1058 
parasitic]) 
1059 
2. Corneal perforation 
1060 
3. Graft detachment ≥50% 
1061 
 
1062 
In addition to the primary safety events, all adverse events (systemic and ocular in donor and 
1063 
recipient eyes) will be captured. The severity of each adverse event and the relationship of the 
1064 
event to the cell therapy procedure will be assessed by an independent Medical Monitor(s) 
1065 
(MM). The independent Medical Monitor’s (see Section 9.3) coding of the adverse event and 
1066 
designation of severity and relatedness to treatment will serve as final to use for adverse event 
1067 
reporting and safety outcome analysis.  
1068 
 
1069 
7.1.2. FEASIBILITY MEASURES 
1070 
Manufacturing feasibility will be evaluated for each biopsy attempt.  At least one CALEC 
1071 
construct will be attempted to be manufactured from each biopsy, as detailed in the MOP. Each 
1072 
attempted construct will undergo Quality Control (QC) testing to determine product conformity to 
1073 
CMCF release criteria per MOP, which includes assessments of cell growth, cell viability, and 
1074 
culture contamination.  If all of these QC release criteria are met for at least one CALEC 
1075 
construct, the biopsy attempt is considered a feasibility success.  
1076 
 
1077 
In the event that a construct met all QC release criteria, but surgery was not performed for a 
1078 
reason unrelated to the development of construct for transplant, the case would still be 
1079 
considered a feasibility success for the biopsy attempt.  
1080 
 
1081 
Up to two biopsy attempts of a single participant may be performed according to Section 5. 
1082 
Feasibility analysis will explore both the number of biopsies per participant and the number of 
1083 
construct attempts per biopsy, and whether those construct attempts met QC release criteria. 
1084 
Each of the individual QC testing and acceptance criteria will be documented to further describe 
1085 
constructs that did not meet the criteria for release.   
1086 
 
1087 
7.2. SECONDARY OUTCOME MEASURES 
1088 
Efficacy Measures: The primary efficacy outcome will be a binary “Complete Success” of the 
1089 
graft, as defined below.   Secondarily, “Partial Success” will also be considered according to a 3 
1090 
category outcome defined below.   
1091 
1. “Complete Success” will be defined as improvement in corneal surface integrity   
1092 
2. “Partial Success” will be defined as 
1093 
a. No improvement in corneal surface integrity and  
1094 
b. Improvement in either 
1095 
i. Extent of corneal vascularization or 
1096 
ii. Participant symptomatology 
1097 
3. Otherwise, not a success 
1098 
 
1099 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 31 of 51 
Improvement in each area is defined as follows, where changes are measured relative to 
1100 
the Baseline visit: 
 
1101 
• 
Corneal surface integrity    
1102 
o If epithelial defect surface area (based on clinical assessment) at the Baseline 
1103 
visit is > 0 mm2, then improvement will be defined as:  
1104 
 
Decrease in epithelial defect surface area (based on clinical assessment 
1105 
by an independent investigator, not the treating surgeon, as described in 
1106 
the MOP) by ≥75% 
1107 
o If epithelial defect surface area (based on clinical assessment) at the Baseline 
1108 
visit is = 0 mm2, then improvement will be defined as:  
1109 
 
Epithelial defect surface area of 0 mm2 and  
1110 
 
Decrease in corneal surface staining (based on clinical assessment by an 
1111 
independent investigator, not the treating surgeon, using NEI grading 
1112 
scale) by ≥50%   
1113 
• 
Extent of corneal vascularization  
1114 
o Decrease in neovascular area (based on digital slit lamp photographs, using 
1115 
mathematical software to calculate as described in the MOP) by ≥25%  
1116 
• 
Participant symptomatology  
1117 
o Decrease in Ocular Surface Disease Index (OSDI) score by ≥25% or 
1118 
o Decrease in Symptom Assessment in Dry Eye (SANDE) score by ≥25%  
1119 
 
1120 
The primary efficacy time points will be at 3 months (to assess early success), as well as 12 and 
1121 
18 months (to assess late success). Efficacy outcomes at all interim post-3 month time points 
1122 
will also be evaluated secondarily. The pattern of efficacy outcomes for an eye at these and all 
1123 
interim time points 3 months and later will be evaluated secondarily to assess the degree of 
1124 
stability of each level of success over time. 
1125 
 
1126 
7.3. OTHER OUTCOME MEASURES 
1127 
In addition to the primary and secondary outcome measures defined above, distribution of data 
1128 
and summary statistics will be evaluated on the following at each time point where relevant data 
1129 
are collected: IOP, impression cytology, corneal opacification, visual acuity, AS-OCT outcomes 
1130 
(central corneal epithelial thickness and limbal epithelial thickness in superior, inferior, nasal, 
1131 
and temporal quadrants), and IVCM outcomes (presence of corneal epithelial cells, presence of 
1132 
conjunctival epithelial cells, and the presence of limbal palisades of Vogt.) All reported adverse 
1133 
events will also be tabulated. 
1134 
 
1135 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 32 of 51 
8. MONITORING STUDY PROGRESS 
1136 
Study progress will be monitored weekly or bi-weekly on Operations Committee calls. 
1137 
Recruitment reports will be available on the study website to monitor recruitment progress and 
1138 
ultimately follow up completion relative to the projected timeline. Operations Committee calls will 
1139 
also serve to keep all units of the Coordinating Center, Clinical Center, and PIs informed and 
1140 
engaged with the day-to-day progress of whether study goals are being met, including 
1141 
successful study oversight (including safety monitoring  and protocol adherence monitoring of 
1142 
the monitoring plan) and eventually plans for closeout, manuscripts, and dissemination of study 
1143 
results. The Operations Committee will also monitor study progress with regards to timeliness 
1144 
and achievement of study goals.  
1145 
 
1146 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 33 of 51 
9. ASSESSMENT OF SAFETY 
1147 
 
1148 
9.1. METHODS AND TIMING FOR ASSESSING, RECORDING, AND REPORTING OF 
1149 
SAFETY PARAMETERS 
1150 
All adverse events will be assessed by investigators and recorded at each visit following the 
1151 
baseline visit. 
1152 
 
1153 
Details of recording and reporting adverse events have been provided in MOP. 
1154 
 
1155 
Details of monitoring and reporting adverse events can be found in the Monitoring Plan.  
1156 
 
1157 
9.2. SAFETY OVERSIGHT  
1158 
Independent Medical Monitors (MMs) will be appointed to this study and will review any reported 
1159 
adverse events on a periodic basis to independently evaluate all adverse events. The MMs will 
1160 
be designated from the EMMES Corporation, Rockville, MD and will be unaffiliated with the 
1161 
CALEC study in any other way. The MMs are responsible for periodic review of all adverse 
1162 
events and within 24 hours of notification for cases that require any expedited reporting (for 
1163 
which an auto-email notification alert at the time of data entry will be generated). Details of the 
1164 
adverse event review by the MM can be found in the Monitoring Plan. The MM coding of the 
1165 
adverse event and designation of severity and relatedness to treatment will serve as final to use 
1166 
for adverse event reporting and safety outcome analysis. 
1167 
 
1168 
The DSMC will be responsible for reviewing the ethical conduct of the study and for monitoring 
1169 
the data for evidence of adverse or beneficial treatment effects.  Adverse events will be reported 
1170 
to the DSMC according to the following: 
1171 
• 
All adverse events will be reviewed with the full DSMC on a periodic basis. 
1172 
• 
A subset of adverse events will be reported expeditiously to designated DSMC members, as 
1173 
defined by the DSMC in the DSMC Charter.     
1174 
• 
During Phase A, designated DSMC members will also be provided with a full report of all 
1175 
adverse events for each Phase A participant who completed CALEC transplant, upon 
1176 
completion of their 2 week visit, to review prior to the consenting and enrolling of the next 
1177 
study participant (or prior to proceeding to a second biopsy for a Phase A participant). 
1178 
• 
Also, if criteria for considering temporary or permanent suspension of the trial are met 
1179 
(Section 9.3), the DSMC will be notified expeditiously. 
1180 
 
1181 
Details of safety oversight by the DSMC can be found in the Monitoring Plan and the DSMC 
1182 
Charter.   
1183 
 
1184 
9.3. STUDY HALTING GUIDELINES 
1185 
The DSMC may recommend at any time whether the study should continue per protocol, be 
1186 
further investigated, be discontinued, or be modified and then proceed. The FDA may also 
1187 
suspend additional enrollment and study interventions/administration of study product for the 
1188 
entire study, if applicable. 
1189 
 
1190 
There will be no formal halting guidelines for safety, as the DSMC will receive full safety reports 
1191 
on Phase A participants as noted in Section 9.2 above, and will also expeditiously receive some 
1192 
events as noted in Section 9.2 above throughout the duration of the study.   
1193 
 
1194 
 
1195 
Halting guidelines for feasibility are as follows:  
1196 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 34 of 51 
• 
A temporary suspension of enrollment will occur if four of the first eight CALEC biopsies, 
1197 
across Phases A and B, result in failure.  
1198 
o This could include two biopsies originating from one participant, (i.e., each biopsy 
1199 
would count separately) 
1200 
o Multiple construct attempts based on the same biopsy would be considered inclusive 
1201 
of a single biopsy 
1202 
• 
If this criterion is met, the DSMC will be notified expeditiously and enrollment activities will 
1203 
be temporarily suspended until further direction from the DSMC. Starting from the time the 
1204 
criteria are identified as met, until the time the DSMC provides further direction, the 
1205 
temporary suspension of enrollment activities will be defined as follows.   
1206 
o No participants will be consented 
1207 
o Participants who have consented and are in some stage of completing screening or 
1208 
baseline visits will be on hold (labs values that have already been processed may be 
1209 
collected and entered into the CRF) 
1210 
o Participants who have not completed a biopsy will be on hold 
1211 
o Participants who had already completed transplant and thus in follow up will not be 
1212 
on hold 
1213 
 
1214 
  
1215 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 35 of 51 
10. MONITORING PROTOCOL ADHERENCE 
1216 
Details for monitoring clinical site for protocol adherence can be found in the Monitoring Plan. 
1217 
The Clinical Center will oversee personnel training and certification, IRB approval and reporting, 
1218 
participant recruitment and retention. The day-to-day local clinical procedures monitoring will be 
1219 
performed by the Clinical Research Supervisor with the support of study website reports 
1220 
provided by the Coordinating Center. The monitoring will be conducted to ensure human 
1221 
participant protection, study procedures, laboratory, study intervention administration, and data 
1222 
collection processes are of high quality and meet ICH E6 and MEEI regulatory guidelines. The 
1223 
Coordinating Center will be responsible for conducting the monitoring visits and ensuring that 
1224 
monitoring findings are reported to the PIs, who will then report to the IRB as necessary and are 
1225 
promptly addressed by the Study Coordinator. 
1226 
 
1227 
The Coordinating Center will provide support via reports and queries for the Clinical Center 
1228 
oversight noted above, including reports for monitoring participant status and visit status 
1229 
(upcoming, pending, past due, missed). In addition, the Coordinating Center will develop and 
1230 
oversee a system for identifying via database queries, documenting, and reporting protocol and 
1231 
procedural deviations to the Clinical Center, PIs, IRB and DSMC. The Coordinating Center will 
1232 
be responsible for conducting on-site monitoring visits with support of the Clinical Center Clinical 
1233 
Research Supervisor. Additional accountability and oversight will be reinforced by Operations 
1234 
Committee monitoring of the activities noted above. This will include reports and review of 
1235 
recruitment, retention, and protocol deviations on at least a monthly basis.   
1236 
 
1237 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 36 of 51 
11. QUALITY CONTROL OF DATA 
1238 
Quality control details can be found in the Monitoring Plan. A major function of the Coordinating 
1239 
Center is to assure that high quality data are collected so that valid analyses can be conducted. 
1240 
The Coordinating Center will collaborate with the Clinical Center and PIs to provide the structure 
1241 
and support to enforce and confirm high quality data are being received. Data are verified on 
1242 
multiple levels across all stages of the study. In brief, this will include development of the 
1243 
following systems, as well as the monitoring and resolution of data issues identified via these 
1244 
systems: 
1245 
1. Source Data / Data Entry Validations: Study data will be collected by completion of an 
1246 
electronic Case Report Form (eCRF), and via paper versions of the case report forms (CRF) 
1247 
as needed. eCRFs and CRFs serve as the primary source document for data collection on 
1248 
study participants and data can be readily entered on the study website directly into the 
1249 
Coordinating Center’s database. Data entry validations include logic and contingency 
1250 
checks at the time of data entry on the website. Validations will also apply to edits made to 
1251 
the eCRFs. Edits are tracked via an audit table in the database. 
1252 
2. Protocol Review: Study data will undergo near real time monitoring in which an automated 
1253 
regular (weekly or monthly) ‘protocol review’ program runs checks including cross form 
1254 
contingencies, write in fields, and data abnormalities, for review and feedback to the Clinical 
1255 
Center. 
1256 
3. Data Cleaning: Data cleaning programs will be run periodically on frozen datasets for DSMC 
1257 
monitoring reports and to be conducted at the end when the database is locked. 
1258 
4. Quality Control Reports: Reports will be generated for use by Coordinating Center, Clinical 
1259 
Center, and for review by oversight committees. 
1260 
 
1261 
Site Monitoring and Protocol Adherence: Quality control of data also includes monitoring of 
1262 
protocol adherence as described in the prior section.  
1263 
 
1264 
 
 
1265 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 37 of 51 
12. CONTINUED FOLLOW UP FOR CLAU PARTICIPANT ENROLLED PRIOR TO 
1266 
PROTOCOL AMENDMENT DISCONTINUING CLAU ARM OBJECTIVE    
1267 
Prior to v5.0 of the CALEC Protocol, a secondary objective of the study was to compare CALEC 
1268 
efficacy and safety measures with the standard treatment alternative, CLAU.  Phase B of the 
1269 
study was designed to randomize participants to receive either CALEC or the standard 
1270 
treatment alternative, CLAU. Due to recruitment challenges, v5.0 amended the Protocol to 
1271 
discontinue the CLAU comparison objective and enroll participants for CALEC treatment only.  
1272 
At the time of the protocol amendment, one participant had been randomized to the CLAU 
1273 
group and completed follow up through at least the 9 month visit.  The prior versions of the 
1274 
protocol describe the biopsy, transplant, and medication regimen procedures that applied to this 
1275 
participant.  The participant will continue to be followed with the remainder of the follow up visits 
1276 
and testing procedures that were required for the CLAU group.        
1277 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 38 of 51 
13. LITERATURE REFERENCES 
1278 
Baradaran-Rafii, A., Ebrahimi, M., Kanavi, M. R., Taghi-Abadi, E., Aghdami, N., Eslani, M., 
1279 
Bakhtiari, P., Einollahi, B., Baharvand, H. & Javadi, M. A. (2010). Midterm outcomes of 
1280 
autologous cultivated limbal stem cell transplantation with or without penetrating 
1281 
keratoplasty. Cornea, 29, 502-9. 
1282 
Basu, S., Ali, H. & Sangwan, V. S. (2012). Clinical Outcomes of Repeat Autologous Cultivated 
1283 
Limbal Epithelial Transplantation for Ocular Surface Burns. Am J Ophthalmol. 
1284 
Clinch, T. E., Goins, K. M. & Cobo, L. M. (1992). Treatment of contact lens-related ocular 
1285 
surface disorders with autologous conjunctival transplantation. Ophthalmology, 99, 634-
1286 
8. 
1287 
Copeland, R. A., Jr. & Char, D. H. (1990). Limbal autograft reconstruction after conjunctival 
1288 
squamous cell carcinoma. Am J Ophthalmol, 110, 412-5. 
1289 
Dietrich-Ntoukas, T., Hofmann-Rummelt, C., Kruse, F. E. & Schlotzer-Schrehardt, U. (2012). 
1290 
Comparative Analysis of the Basement Membrane Composition of the Human Limbus 
1291 
Epithelium and Amniotic Membrane Epithelium. Cornea. 
1292 
Dua, H. S. & Azuara-Blanco, A. (1999). Allo-limbal transplantation in patients with limbal stem 
1293 
cell deficiency. Br J Ophthalmol, 83, 414-9. 
1294 
Dua, H. S. & Forrester, J. V. (1990). The corneoscleral limbus in human corneal epithelial 
1295 
wound healing. Am J Ophthalmol, 110, 646-56. 
1296 
Dua, H. S., Miri, A. & Said, D. G. (2010). Contemporary limbal stem cell transplantation - a 
1297 
review. Clin Experiment Ophthalmol, 38, 104-17. 
1298 
Fernandes, M., Sridhar, M. S., Sangwan, V. S. & Rao, G. N. (2005). Amniotic membrane 
1299 
transplantation for ocular surface reconstruction. Cornea, 24, 643-53. 
1300 
Fogla, R. & Padmanabhan, P. (2005). Deep anterior lamellar keratoplasty combined with 
1301 
autologous limbal stem cell transplantation in unilateral severe chemical injury. Cornea, 
1302 
24, 421-5. 
1303 
Grueterich, M., Espana, E. M. & Tseng, S. C. (2003). Ex vivo expansion of limbal epithelial stem 
1304 
cells: amniotic membrane serving as a stem cell niche. Surv Ophthalmol, 48, 631-46. 
1305 
Haamann, P., Jensen, O. M. & Schmidt, P. (1998). Limbal autograft transplantation. Acta 
1306 
Ophthalmol Scand, 76, 117-8. 
1307 
Holland, E. J. & Schwartz, G. S. (1996). The evolution of epithelial transplantation for severe 
1308 
ocular surface disease and a proposed classification system. Cornea, 15, 549-56. 
1309 
Holland, E. J. & Schwartz, G. S. (1999). Epithelial stem-cell transplantation for severe ocular-
1310 
surface disease. The New England journal of medicine, 340, 1752-3. 
1311 
Holland, E. J. & Schwartz, G. S. (2000). Changing concepts in the management of severe 
1312 
ocular surface disease over twenty-five years. Cornea, 19, 688-98. 
1313 
Jenkins, C., Tuft, S., Liu, C. & Buckley, R. (1993). Limbal transplantation in the management of 
1314 
chronic contact-lens-associated epitheliopathy. Eye (Lond), 7 ( Pt 5), 629-33. 
1315 
Kenyon, K. R. & Tseng, S. C. (1989). Limbal autograft transplantation for ocular surface 
1316 
disorders. Ophthalmology, 96, 709-22; discussion 722-3. 
1317 
Koizumi, N., Inatomi, T., Quantock, A. J., Fullwood, N. J., Dota, A. & Kinoshita, S. (2000). 
1318 
Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for 
1319 
autologous transplantation in rabbits. Cornea, 19, 65-71. 
1320 
Koizumi, N., Inatomi, T., Suzuki, T., Sotozono, C. & Kinoshita, S. (2001). Cultivated corneal 
1321 
epithelial stem cell transplantation in ocular surface disorders. Ophthalmology, 108, 
1322 
1569-74. 
1323 
Kolli, S., Ahmad, S., Lako, M. & Figueiredo, F. (2010). Successful clinical implementation of 
1324 
corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. 
1325 
Stem cells, 28, 597-610. 
1326 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 39 of 51 
Kruse, F. E., Chen, J. J., Tsai, R. J. & Tseng, S. C. (1990). Conjunctival transdifferentiation is 
1327 
due to the incomplete removal of limbal basal epithelium. Invest Ophthalmol Vis Sci, 31, 
1328 
1903-13. 
1329 
Lee, S. H. & Tseng, S. C. (1997). Amniotic membrane transplantation for persistent epithelial 
1330 
defects with ulceration. Am J Ophthalmol, 123, 303-12. 
1331 
Morgan, S. & Murray, A. (1996). Limbal autotransplantation in the acute and chronic phases of 
1332 
severe chemical injuries. Eye (Lond), 10 ( Pt 3), 349-54. 
1333 
Nakamura, T., Inatomi, T., Sotozono, C., Ang, L. P., Koizumi, N., Yokoi, N. & Kinoshita, S. 
1334 
(2006). Transplantation of autologous serum-derived cultivated corneal epithelial 
1335 
equivalents for the treatment of severe ocular surface disease. Ophthalmology, 113, 
1336 
1765-72. 
1337 
Nakamura, T., Koizumi, N., Tsuzuki, M., Inoki, K., Sano, Y., Sotozono, C. & Kinoshita, S. 
1338 
(2003). Successful regrafting of cultivated corneal epithelium using amniotic membrane 
1339 
as a carrier in severe ocular surface disease. Cornea, 22, 70-1. 
1340 
Nishida, K., Yamato, M., Hayashida, Y., Watanabe, K., Yamamoto, K., Adachi, E., Nagai, S., 
1341 
Kikuchi, A., Maeda, N., Watanabe, H., Okano, T. & Tano, Y. (2004). Corneal 
1342 
reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal 
1343 
epithelium. N Engl J Med, 351, 1187-96. 
1344 
Pellegrini, G., Traverso, C. E., Franzi, A. T., Zingirian, M., Cancedda, R. & De Luca, M. (1997). 
1345 
Long-term restoration of damaged corneal surfaces with autologous cultivated corneal 
1346 
epithelium. Lancet, 349, 990-3. 
1347 
Prabhasawat, P., Kosrirukvongs, P., Booranapong, W. & Vajaradul, Y. (2001). Amniotic 
1348 
membrane transplantation for ocular surface reconstruction. J Med Assoc Thai, 84, 705-
1349 
18. 
1350 
Puangsricharern, V. & Tseng, S. C. (1995). Cytologic evidence of corneal diseases with limbal 
1351 
stem cell deficiency. Ophthalmology, 102, 1476-85. 
1352 
Rama, P., Matuska, S., Paganoni, G., Spinelli, A., De Luca, M. & Pellegrini, G. (2010). Limbal 
1353 
stem-cell therapy and long-term corneal regeneration. N Engl J Med, 363, 147-55. 
1354 
Resnikoff, S., Pascolini, D., Mariotti, S. P. & Pokharel, G. P. (2008). Global magnitude of visual 
1355 
impairment caused by uncorrected refractive errors in 2004. Bulletin of the World Health 
1356 
Organization, 86, 63-70. 
1357 
Sangwan, V. S., Matalia, H. P., Vemuganti, G. K., Ifthekar, G., Fatima, A., Singh, S. & Rao, G. 
1358 
N. (2005). Early results of penetrating keratoplasty after cultivated limbal epithelium 
1359 
transplantation. Arch Ophthalmol, 123, 334-40. 
1360 
Schermer, A., Galvin, S. & Sun, T. T. (1986). Differentiation-related expression of a major 64K 
1361 
corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem 
1362 
cells. J Cell Biol, 103, 49-62. 
1363 
Schwab, I. R., Reyes, M. & Isseroff, R. R. (2000). Successful transplantation of bioengineered 
1364 
tissue replacements in patients with ocular surface disease. Cornea, 19, 421-6. 
1365 
Shahdadfar, A., Haug, K., Pathak, M., Drolsum, L., Olstad, O. K., Johnsen, E. O., Petrovski, G., 
1366 
Moe, M. C. & Nicolaissen, B. (2012). Ex vivo expanded autologous limbal epithelial cells 
1367 
on amniotic membrane using a culture medium with human serum as single supplement. 
1368 
Exp Eye Res, 97, 1-9. 
1369 
Shimazaki, J., Aiba, M., Goto, E., Kato, N., Shimmura, S. & Tsubota, K. (2002). Transplantation 
1370 
of human limbal epithelium cultivated on amniotic membrane for the treatment of severe 
1371 
ocular surface disorders. Ophthalmology, 109, 1285-90. 
1372 
Shimazaki, J., Higa, K., Morito, F., Dogru, M., Kawakita, T., Satake, Y., Shimmura, S. & 
1373 
Tsubota, K. (2007). Factors influencing outcomes in cultivated limbal epithelial 
1374 
transplantation for chronic cicatricial ocular surface disorders. Am J Ophthalmol, 143, 
1375 
945-53. 
1376 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 40 of 51 
Shortt, A. J., Secker, G. A., Notara, M. D., Limb, G. A., Khaw, P. T., Tuft, S. J. & Daniels, J. T. 
1377 
(2007). Transplantation of ex vivo cultured limbal epithelial stem cells: a review of 
1378 
techniques and clinical results. Surv Ophthalmol, 52, 483-502. 
1379 
Smolin, G. a. T., Richard A. 2005. Smolin and Thoft’s The cornea : scientific foundations and 
1380 
clinical practice, Philadelphia : Lippincott Williams & Wilkins. 
1381 
Tan, D. T., Ficker, L. A. & Buckley, R. J. (1996). Limbal transplantation. Ophthalmology, 103, 
1382 
29-36. 
1383 
Tsai, R. J. & Tsai, R. Y. (2010). Ex vivo expansion of corneal stem cells on amniotic membrane 
1384 
and their outcome. Eye Contact Lens, 36, 305-9. 
1385 
Tsai, R. J. & Tseng, S. C. (1988). Substrate modulation of cultured rabbit conjunctival epithelial 
1386 
cell differentiation and morphology. Invest Ophthalmol Vis Sci, 29, 1565-76. 
1387 
Tseng, S. C. (1989). Concept and application of limbal stem cells. Eye (Lond), 3 ( Pt 2), 141-57. 
1388 
Tseng, S. C. (1996). Regulation and clinical implications of corneal epithelial stem cells. Mol Biol 
1389 
Rep, 23, 47-58. 
1390 
Tseng, S. C., Kruse, F. E., Merritt, J. & Li, D. Q. (1996). Comparison between serum-free and 
1391 
fibroblast-cocultured single-cell clonal culture systems: evidence showing that epithelial 
1392 
anti-apoptotic activity is present in 3T3 fibroblast-conditioned media. Curr Eye Res, 15, 
1393 
973-84. 
1394 
Tseng, S. C., Meller, D., Anderson, D. F., Touhami, A., Pires, R. T., Gruterich, M., Solomon, A., 
1395 
Espana, E., Sandoval, H., Ti, S. E. & Goto, E. (2002). Ex vivo preservation and 
1396 
expansion of human limbal epithelial stem cells on amniotic membrane for treating 
1397 
corneal diseases with total limbal stem cell deficiency. Adv Exp Med Biol, 506, 1323-34. 
1398 
Yalcindag, F. N., Incel, O. & Ozdemir, O. (2008). Effectiveness of tacrolimus in high-risk limbal 
1399 
allo-graft transplantation. Ann Ophthalmol (Skokie), 40, 152-6. 
1400 
Yao, Y. F., Zhang, B., Zhou, P. & Jiang, J. K. (2002). Autologous limbal grafting combined with 
1401 
deep lamellar keratoplasty in unilateral eye with severe chemical or thermal burn at late 
1402 
stage. Ophthalmology, 109, 2011-7. 
1403 
Zakaria, N., Koppen, C., Van Tendeloo, V., Berneman, Z., Hopkinson, A. & Tassignon, M. J. 
1404 
(2010). Standardized limbal epithelial stem cell graft generation and transplantation. 
1405 
Tissue Eng Part C Methods, 16, 921-7. 
1406 
 
1407 
 
1408 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 41 of 51 
14. APPENDICES 
1409 
14.1. APPENDIX I: SYMPTOM MEASUREMENT SCALES 
1410 
 
1411 
Ocular Surface Disease Index (OSDI) (Vitale et al., 2004) 
1412 
 
1413 
 
1414 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 42 of 51 
 
1415 
 
 
1416 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 43 of 51 
 
1417 
Symptom Assessment iN Dry Eye (SANDE) Questionnaire 
1418 
(Schaumberg et al., 2007) 
1419 
 
1420 
 
1421 
 
1422 
 
1423 
Please complete the following questions regarding the frequency and severity of your 
1424 
dry eye symptoms. 
1425 
 
1426 
 
1427 
1. Frequency of symptoms:  
1428 
 
1429 
Please place an ‘X’ on the line to indicate how often, on average, your eyes feel dry and/or 
1430 
irritated:  
1431 
 
1432 
_______________________________________________ 
1433 
Rarely                                                            All of the Time 
1434 
 
1435 
 
1436 
 
1437 
2. Severity of symptoms:  
1438 
 
1439 
Please place an ‘X’ on the line to indicate how severe, on average, your eyes feel dry and/or 
1440 
irritated:  
1441 
 
1442 
_______________________________________________ 
1443 
Very Mild                                                       Very Severe 
1444 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 44 of 51 
14.2. APPENDIX II: NATIONAL EYE INSTITUTE CORNEAL FLUORESCEIN GRADING 
1445 
SCALE 
1446 
 
1447 
 
1448 
 
1449 
 
1450 
 
1451 
For corneal fluorescein staining, saline-moistened fluorescein strips or 1% sodium fluorescein 
1452 
solution will be used to stain the tear film. The entire cornea will then be examined using slit 
1453 
lamp evaluation with a yellow barrier filter (#12 Wratten) and cobalt blue illumination.  Each of 
1454 
five corneal zones (superior, nasal, central, inferior, and temporal) will be graded from 0 
1455 
(normal) to 3 (severe) and total staining score of the cornea will be calculated by adding the 
1456 
scores of all 5 zones together. The scores will range r from 0 to 15 points. 
1457 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 45 of 51 
14.3. APPENDIX III: VASCULARIZATION MEASUREMENT TOOL 
1458 
Digital Quantification of Neovascularization 
1459 
 
1460 
 
1461 
 
1462 
Digital slit lamp corneal images are analyzed using graphics-editing software (Photoshop) and a 
1463 
mathematical program (Java script from ImageJ).  After the total area is delineated, the blood 
1464 
vessels are isolated using Photoshop.  Neovascular area is computed using ImageJ to measure 
1465 
the area of the vessels in pixels. 
1466 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 46 of 51 
14.4. APPENDIX IV: FANTES SCALE FOR CORNEAL OPACITY 
1467 
 
1468 
Grade 
Description 
0 
Totally clear such that no opacity could be seen by any method of slit lamp 
microscopic examination 
0.5 
A trace or a faint corneal haze seen only by indirect broad tangential illumination 
1 
Haze of minimal density seen with difficulty with direct and diffuse illumination 
2 
A mild haze easily visible with direct focal slit illumination 
3 
A moderately dense opacity that partially obscured the iris details 
4 
A severely dense opacity that obscured completely the details of intraocular 
structures 
1469 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
Page 47 of 51 
14.5. APPENDIX V: IMPRESSION CYTOLOGY 
1470 
 
1471 
 
1472 
 
1473 
 
1474 
 
1475 
 
1476 
 
1477 
Reporting: 
1478 
 
1479 
Region 
Goblet Cells 
Cornea 
Superior 
 
Inferior 
 
Temporal 
 
Nasal 
 
1480 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
 
 
 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
 
 
 
 
 
Page 48 of 51 
1 
14.6. APPENDIX VI: SCHEDULES OF EVENTS & PROCEDURES FOR PARTICIPANTS COMPLETING TRANSPLANT 
2 
 
3 
 
4 
 
5 
Key:  X=Donor Eye; X=Recipient Eye; Eye procedures marked with a black X will be performed on both the recipient and donor eyes (or the participant). 
1Perform only if needed and deemed appropriate by clinician. 2Needed only if MRSA was previously detected. 3Baseline procedures may be conducted over multiple visits after screening. 4Portions of exam/photo that require staining in recipient eye are at investigator’s discretion. 5The 
2 week, 9 month, and 15 month visits will be optional for participants for whom travel is prohibitive (see Protocol Section 6.8 for more details).   
 
Visit 
1 
2 
3 
4 
Transportation of Tissue to GMP lab and Preparation of Cell Sheets 
5 
6 
Transportation of CALEC to Mass. Eye and Ear– Release anticipated 10 to 30 days after biopsy 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Time Period & Procedure 
(post-transplant visits timed 
from transplant date) 
Screening Baseline3 
Blood draw collected within 1-7 days before or after biopsy 
Limbal Biopsy (Within 25 days of Baseline) 
Day 1 
Post-
Biopsy 
Pre-Op 
Corneal Reconstruction with CALEC (Day 0) – Within 24 hours of release 
Day 1 
Post-
Graft 
Week 1 Week 25 Month 1 
(4 weeks) 
Month 3  
(13 weeks) 
Month 6 
(26 weeks) 
Month 95 
(39 weeks) 
Month 12 
 (52 weeks) 
Month 155 
(65 weeks) 
Month 18 
(78 weeks) 
Timing Window  
 
Within 30 
days of 
Screening 
1-2 days 
after 
biopsy 
1 to 5 
days prior 
to transpl. 
±0 days ± 3 days ± 3 days ± 3 days ± 1 week 
± 1 
week 
± 1 week ± 1 week ± 2 week ± 2 week 
Obtain Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility Assessment 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
Past Medical History 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
Pre-Op Health Screening 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematologic Lab Screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy Test 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Slit Lamp Examination 
X 
X 
X 
X 
X4 
X4 
X4 
X 
X 
X 
X 
X 
X 
X 
Visual Acuity BCVA 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Intraocular Pressure  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Fundus Examination or B-
Scan Ultrasound 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Schirmer’s Test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
AS-OCT 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
X 
IVCM 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
X 
Slit Lamp Photos  
X 
X 
X 
 
X4 
X4 
X4 
X 
X 
X 
X 
X 
X 
X 
Conjunctival Swab & Culture 
X 
 
 
 
 
 
 
 
X2 
 
 
X2 
 
 
Impression Cytology1 
 
 
 
 
 
 
 
 
 
 
 
X1 
X1 
X1 
Punctal Plugs1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Bandage Contact Lens1 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Antibiotic 
 
 
X 
X 
X 
X 
X 
X 
X1 
X1 
X1 
X1 
X1 
X1 
Topical steroid and 
fluoroquinolone 
 
 
X 
X 
X 
X 
X 
X 
X1 
X1 
X1 
X1 
X1 
X1 
Vancomycin2 
 
X 
X 
X 
X 
X 
X 
X 
X1 
X1 
X1 
X1 
X1 
X1 
Artificial Tears 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
20% Autologous Serum Drops 
 
 
 
 
X 
X 
X 
X1 
X1 
X1 
X1 
X1 
X1 
X1 
Symptom Assessment 
 
X 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
Adverse Event Assessment 
 
X (changes 
in medical 
status) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
 
 
 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
 
 
 
 
 
Page 49 of 51 
 
1486 
 
1487 
15. PROTOCOL ADDENDUM: SECOND TRANSPLANT   
1488 
 
1489 
15.1. OVERVIEW   
1490 
Some study participants who received CALEC and for whom epithelial defect and conjunctivalization 
1491 
has not resolved after 12 months may benefit from an additional transplant.  Participants meeting study 
1492 
criteria (section 15.3) may be offered the opportunity to undergo a second procedure, either CALEC or 
1493 
CLAU.  The following chapter outlines the protocol that will be followed for these participants. Up to 
1494 
three participants can receive a second transplant. 
1495 
 
1496 
15.2. CRITERIA FOR SECOND TRANSPLANT 
1497 
All of the following criteria must be met in order for a participant to be considered for a second 
1498 
transplant. 
1499 
• Participant previously received CALEC (participants previously receiving CLAU will not be 
1500 
eligible) 
1501 
• Second transplant cannot occur prior to the 12-month visit 
1502 
• Presence of epithelial defect in recipient eye 
1503 
• Must have been present at 2 or more visits (>1 month between visits), any time on or after 
1504 
the 3-month visit  
1505 
• >1 mm in any dimension 
1506 
• (Note: Does not need to be present at the Screening/Baseline visit prior to second 
1507 
transplant) 
1508 
• Presence of conjunctivalization / fibrovascular pannus in recipient eye 
1509 
• Must be present at the Screening/Baseline visit prior to second transplant  
1510 
• >2 mm from the limbus  
1511 
• ≥6 clock hours 
1512 
• Investigator discretion that repeat transplant has therapeutic potential 
1513 
• Absence of the following in the donor eye:  
1514 
• Conjunctivalization of the cornea defined by fibrovascular pannus more than 2mm from 
1515 
the limbus into the cornea for ≥3 clock hours 
1516 
• Lack of limbal palisades of Vogt for ≥3 clock hours 
1517 
• History of allo-limbal transplantation 
1518 
 
1519 
NOTE: these criteria are independent of whether the participant meets the protocol definition of 
1520 
complete or partial success. 
1521 
 
1522 
15.3. INFORMED CONSENT PROCESS 
1523 
Participants meeting criteria and offered a second transplant option will undergo an informed consent 
1524 
process.  The informed consent process will include a discussion with participants that they have the 
1525 
option of CLAU, CALEC, or no second transplant. 
1526 
 
1527 
15.4. SCREENING AND BASELINE  
1528 
Participants consenting to undergo a second transplant, either CLAU or CALEC, will undergo the same 
1529 
screening and baseline procedures in the protocol for the initial CALEC transplant (section 6.8.1-6.8.2), 
1530 
with the following exceptions:   
1531 
• The screening visit will be combined with baseline visit since the subjects receiving the second 
1532 
graft will have already been followed for at least a year since their initial CALEC transplant  
1533 
• The combined screening/baseline visit will include the following:  
1534 
o Evaluation of eligibility for a second graft  
1535 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
 
 
 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
 
 
 
 
 
Page 50 of 51 
o Hematologic lab screening will include only the blood draw collected within 1-7 days as 
1536 
required for manufacturing of the graft per DCDI protocol  
1537 
 Hepatitis B   
1538 
 Hepatitis C  
1539 
 HIV  
1540 
o Slit lamp exam  
1541 
o BCVA 
1542 
o IOP 
1543 
o Fundus exam or B-scan 
1544 
o AS-OCT  
1545 
o IVCM  
1546 
o Slit lamp photos 
1547 
o Symptom assessment 
1548 
o Conjunctival swab and culture   
1549 
 (if MRSA positive, the original protocol will be followed for vancomycin) 
1550 
o Schirmer’s test (optional) 
1551 
o Punctal plus (optional) 
1552 
The visit will not require punctal plugs, bandage contact lenses, prednisolone, vancomycin, 
1553 
preservative-free tears and autologous  
1554 
serum tears as participants will already be using them as treatments from initial CALEC. The 
1555 
preoperative antibiotic drop will be given prior to the biopsy and transplant in the respective operative 
1556 
eyes, as with the initial CALEC (section 6.2.1). The Schirmer test may not be needed as participants 
1557 
would have had it to receive the initial CALEC and would have had plugs if necessary.  . The full set of 
1558 
labs will not be repeated as they would have been done to enter the study for the initial CALEC. The 
1559 
labs that are required for manufacturing of CALEC will be repeated as noted above, for participants 
1560 
pursuing second CALEC transplant.    
1561 
 
1562 
15.5. BIOPSY AND TRANSPLANT  
1563 
 
1564 
For participants choosing to undergo CLAU 
1565 
 The pre-operative visit will occur within 60 days after baseline and within 1-5 days prior to 
1566 
transplant, and testing will include slit lamp exam, visual acuity, IOP. 
1567 
 The biopsy and transplant procedures and subsequent medication regimens will follow standard 
1568 
care.   
1569 
 
1570 
For participants choosing to undergo CALEC 
1571 
 The post-biopsy and pre-operative visits and testing schedule, including all procedures and 
1572 
measurements, will be the same as the current protocol (APPENDIX VI).  
1573 
 The CALEC manufacturing (section 6.1), biopsy and transplant procedures (section 6.2) and 
1574 
subsequent medication regimens (section 6.5) will follow the protocol for initial CALEC 
1575 
 If a participant experiences failure of the biopsy to generate a viable cell sheet, the participant 
1576 
will not be eligible to rescreen for another biopsy attempt.  The participant will be monitored by 
1577 
the study team for 30 days to check for any adverse events related to the biopsy procedures.   If 
1578 
the donor eye has a complication or adverse event that does not resolve after 30 days, the study 
1579 
team will continue to monitor until it resolves.  The participant will discontinue the study after the 
1580 
monitoring period.  
1581 

Safety and Feasibility of Cultivated Autologous Limbal Epithelial 
 
 
 
 
 
 
 
 
 
PI: Ula Jurkunas, MD 
Cell Transplantation in the Treatment of Limbal Stem Cell Deficiency 
CALEC Protocol_Feb 8 2022 _v6.0 
 
 
 
 
 
 
Page 51 of 51 
  
1582 
 
1583 
15.6. STUDY PROCEDURES, MEASUREMENTS, AND VISIT SCHEDULE 
1584 
 
1585 
For all participants choosing to undergo a second transplant (either CALEC or CLAU), the post-
1586 
operative visit and testing schedule, including all procedures, measurements and data collection, will be 
1587 
the same as the current protocol (APPENDIX VI).  The protocol schedule will be followed until a 
1588 
common study closeout date is reached.       
1589 
 
1590 
15.7. ANALYSIS CONSIDERATIONS 
1591 
15.7.1. SAFETY 
1592 
 Safety events of interest, as defined in section 7.1.1.:    
1593 
o Primary safety analysis for protocol objective:  Safety events will only be tabulated up 
1594 
to (not including) the date of the biopsy for the second CALEC or CLAU transplant. 
1595 
o Additional analysis evaluating second CALEC transplant: Safety events following the 
1596 
biopsy for the second CALEC transplant will be summarized separately.   
1597 
 
1598 
15.7.2. FEASIBILITY 
1599 
 Feasibility measures, as defined in section 7.1.2.:   
1600 
o Primary feasibility analysis for protocol objective: The biopsy attempts for only the 
1601 
initial CALEC transplants will be included in the primary feasibility outcome calculation of 
1602 
feasibility success  
1603 
o Additional analysis evaluating second CALEC transplant: The biopsy attempts for 
1604 
second CALEC transplant will be summarized separately  
1605 
  
1606 
15.7.3. EFFICACY 
1607 
 Efficacy outcome measures, as defined in section 7.2, will be evaluated the following 
1608 
ways: 
1609 
o Primary efficacy analysis for protocol objective:  Efficacy outcome data will only be 
1610 
included up to (not including) the date of biopsy for the second CALEC or CLAU 
1611 
transplant 
1612 
o Additional analyses evaluating second CALEC transplant:  
1613 
 Efficacy outcome data will be evaluated relative to original baseline through the 
1614 
latest protocol visit prior to the study closeout date.  
1615 
 Efficacy outcome data will be evaluated relative to second CALEC baseline 
1616 
through the latest protocol visit prior to the study closeout date.  
1617 
 
1618